Cell culture medium for adamts protein expression

ABSTRACT

The present invention provides culture mediums that are useful for the expression of ADAMTS proteins, such as ADAMTS13. Methods for the expression and purification of ADAMTS proteins are also provided. In some embodiments, the mediums and methods of the invention are useful for the expression of ADAMTS proteins having high specific activities. Also provided are ADAMTS, e.g., ADAMTS13, protein compositions with high specific activities, which are expressed and purified according to the methods provided herein.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/232,626, filed Aug. 9, 2016, which is a continuation of U.S.patent application Ser. No. 14/807,745, filed Jul. 23, 2015, which is acontinuation of U.S. patent application Ser. No. 14/276,819, filed May13, 2014, which is a continutation of U.S. patent application Ser. No.13/622,939, filed Sep. 19, 2012 (now issued as U.S. Pat. No. 8,759,026),which is a continuation of U.S. patent application Ser. No. 12/847,999,filed Jul. 30, 2010 (now issued as U.S. Pat. No. 8,313,926), and claimsthe benefit of U.S. Provisional Application No. 61/230,477 filed Jul.31, 2009, which are all expressly incorporated herein by reference inits entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

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REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

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BACKGROUND OF THE INVENTION

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin typeI motifs) proteins are a family of metalloproteinases containing anumber of conserved domains, including a zinc-dependant catalyticdomain, a cystein-rich domain, a disintegrin-like domain, and at leastone, and in most cases multiple, thrombospondin type I repeats (forreview, see Nicholson et al., BMC Evol Biol. 2005 Feb. 4; 5(1):11).These proteins, which are evolutionarily related to the ADAM and MMPfamilies of metalloproteinases (Jones G C, Curr Pharm Biotechnol. 2006February; 7(1):25-31), are secreted enzymes that have been linked to anumber of diseases and conditions including thrombotic thrombocytopenicpurpura (TTP) (Moake J L, Semin Hematol. 2004 January; 41(1):4-14),connective tissue disorders, cancers, inflammation (Nicholson et al.),and severe plasmodium falciparum malaria (Larkin et al., PLoS Pathog.2009 March; 5(3):e1000349). Because of these associations, the ADAMTSenzymes have been recognized as potential therapeutic targets for anumber of pathologies (Jones G C, Curr Pharm Biotechnol. 2006 February;7(1):25-31). Accordingly, methods of producing large yields of ADAMTSproteins having high specific activities, which are free of contaminantssuch as viruses, BSE, and pathogens like Mycoplasma bacteria, areneeded.

For cultivation of cells, particularly eukaryotic cells, and morespecifically mammalian cells, there is a constant need to use specialculture media providing nutrient substances that are required forefficient growth of cells and for the production of biological products,especially biopharmaceuticals, such as, for example, recombinantproteins, antibodies, viruses, viral antigens, and virus-like particles.For the efficient production of said biological products, it isimportant to achieve an optimal cell density as well as an increase ofthe protein expression itself in order to obtain maximal product yield.

Cell culture media formulations have been supplemented with a range ofadditives, including undefined components like fetal calf serum (FCS),several animal derived proteins and/or protein hydrolysates of bovineorigin as well as protein hydrolysates derived from plants or yeast.

In general, serum or serum-derived substances, such as, e.g., albumin,transferrin or insulin, may comprise unwanted agents that cancontaminate the cell cultures and the biological products obtainedthereof. Furthermore, human serum derived additives have to be testedfor all known viruses, including hepatitis viruses and HIV which can betransmitted via serum. Moreover, bovine serum and products derivedthereof bear the risk of BSE contamination. In addition, allserum-derived products can be contaminated by unknown substances. Whenusing serum or protein additives derived from human or animal sources incell culture, there are numerous problems (e.g., the varying quality incomposition of different batches and the risk of contamination withmycoplasma, viruses or BSE), particularly if the cells are used in themanufacture of drugs or vaccines for human administration.

Therefore, many attempts have been made to provide efficient hostsystems and cultivation conditions, which do not require serum or otheranimal protein compounds.

Such serum-free media have been developed on the basis of proteinextracts derived from plants or yeast. For example, soy hydrolysates areknown to be useful for fermentation processes and can enhance the growthof many fastidious organisms, yeasts and fungi. WO 96/26266 describesthat papaic digests of soy meal are a source of carbohydrate andnitrogen and many of the components can be used in tissue culture.Franek et al. (Biotechnology Progress (2000) 16, 688-692) describegrowth and productivity promoting effects of defined soy and wheathydrolysate peptide fractions.

WO 96/15231 discloses a serum-free medium composed of a syntheticminimal essential medium and a yeast extract for the propagation ofvertebrate cells and a virus production process. A medium formulationcomposed of a basal cell culture medium comprising a rice peptide and anextract of yeast and an enzymatic digest thereof, and/or a plant lipidfor growth of animal cells is disclosed in WO 98/15614. A mediumcomprising purified soy hydrolysate for the cultivation of recombinantcells is disclosed in WO 01/23527. WO 00/03000 discloses a medium thatcomprises a soy hydrolysate and a yeast extract, but also requires thepresence of recombinant forms of animal proteins, such as growthfactors.

EP-A-0 481 791 describes a biochemically defined culture medium forculturing engineered CHO cells, which is free from protein, lipid andcarbohydrate isolated from an animal source, further comprising arecombinant insulin or insulin analogue, 1% to 0.025% w/v papaindigested soy peptone and putrescine. WO 98/08934 describes a serum-freeeukaryotic cell culture comprising hydrolyzed soy peptides (1-1000mg/L), 0.01 to 1 mg/L putrescine and a variety of animal-derivedcomponents, including albumin, fetuin, various hormones and otherproteins. In this context, it should be noted that putrescine is alsoknown to be comprised in standard media like DMEM/Ham's F12 in aconcentration of 0.08 mg/L.

The plant and/or yeast hydrolysates, however, are undefined mixtures ofoligopeptides and other unknown components and contaminants. Moreover,the quality of commercially available lots of hydrolysates variesextremely. As a result, there are large variations in the production ofrecombinant proteins or viral products (a variation of up to a factor ofthree) as a function of the lots of hydrolysates used (“lot-to-lotvariation”). This drawback affects the proliferation of the cells aswell as the protein expression of each cell. US 2007/0212770 describesvarious animal protein-free and oligopeptide-free, chemically definedculture mediums that are useful for the large-scale production ofrecombinant protein biopharmaceuticals.

One ADAMTS family member, ADAMTS13, cleaves von Willebrand factor (vWF)between residues Tyr 1605 and Met 1606, a function responsible for thedegradation of large vWF multimers in vivo. Loss of ADAMTS13 activityhas been linked to a number of conditions, such as TTP (Moake J L, SeminHematol. 2004 January; 41(1):4-14), acute and chronic inflammation(Chauhan et al., J Exp Med. 2008 Sep. 1; 205(9):2065-74), and mostrecently, severe plasmodium falciparum malaria (Larkin et al., PLoSPathog. 2009 March; 5(3):e1000349).

Thrombotic thrombocytopenic purpura (TTP) is a disorder characterized bythrombotic microangiopathy, thrombocytopenia and microvascularthrombosis that can cause various degrees of tissue ischemia andinfarction. Clinically, TTP patients are diagnosed by symptoms such asthrombocytopenia, schistocytes (fragments of erythrocytes) and elevatedlevels of lactate dehydrogenase (Moake J L. Thromboticmicroangiopathies. N Engl J Med. 2002; 347:589-600; Moake J L. vonWillebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura.Semin Hematol. 2004; 41:4-14; Sadler J E, Moake J L, Miyata T, George JN. Recent advances in thrombotic thrombocytopenic purpura. Hematology(Am Soc Hematol Educ Program). 2004: 407-423; Sadler J E. New conceptsin von Willebrand disease. Annu Rev Med. 2005; 56:173-191).

In 1982, Moake et al. found unusually large von Willebrand factor(UL-vWF) multimers in the plasma of the patients with chronic relapsingTTP (Moake J L, Rudy C K, Troll J H, Weinstein M J, Colannino N M,Azocar J, Seder R H, Hong S L, Deykin D. Unusually large plasma factorVIII:von Willebrand factor multimers in chronic relapsing thromboticthrombocytopenic purpura. N Engl J Med. 1982; 307:1432-1435). The linkbetween UL-vWF and TTP gained support with independent findings byFurlan et al. and Tsai and Lian that most patients suffering from TTPare deficient in a plasma metalloprotease, now known to be ADAMTS13,that cleaves vWF (Furlan M, Robles R, Solenthaler M, Wassmer M, SandozP, Laemmle B. Deficient activity of von Willebrand factor-cleavingprotease in chronic relapsing thrombotic thrombocytopenic purpura.Blood. 1997; 89:3097-3103; Tsai H M, Sussman, I I, Ginsburg D, LankhofH, Sixma J J, Nagel R L. Proteolytic cleavage of recombinant type 2A vonWillebrand factor mutants R834W and R834Q: inhibition by doxycycline andby monoclonal antibody VP-1. Blood. 1997; 89:1954-1962; Tsai H M, Lian EC. Antibodies to von Willebrand factor-cleaving protease in acutethrombotic thrombocytopenic purpura. N Engl J Med. 1998; 339:1585-1594).

The ADAMTS13 protease is a 190 kDa glycosylated protein producedpredominantly by the liver (Levy G G, Nichols W C, Lian E C, Foroud T,McClintick J N, McGee B M, Yang A Y, Siemieniak D R, Stark K R, GruppoR, Sarode R, Shurin S B, Chandrasekaran V, Stabler S P, Sabio H,Bouhassira E E, Upshaw J D, Jr., Ginsburg D, Tsai H M. Mutations in amember of the ADAMTS gene family cause thrombotic thrombocytopenicpurpura. Nature. 2001; 413:488-494; Fujikawa K, Suzuki H, McMullen B,Chung D. Purification of human von Willebrand factor-cleaving proteaseand its identification as a new member of the metalloproteinase family.Blood. 2001; 98:1662-1666; Zheng X, Chung D, Takayama T K, Majerus E M,Sadler J E, Fujikawa K. Structure of von Willebrand factor-cleavingprotease (ADAMTS13), a metalloprotease involved in thromboticthrombocytopenic purpura. J Biol Chem. 2001; 276:41059-41063; Soejima K,Mimura N, Hirashima M, Maeda H, Hamamoto T, Nakagaki T, Nozaki C. Anovel human metalloprotease synthesized in the liver and secreted intothe blood: possibly, the von Willebrand factor-cleaving protease; JBiochem (Tokyo). 2001; 130:475-480; Gerritsen H E, Robles R, Lammle B,Furlan M. Partial amino acid sequence of purified von Willebrandfactor-cleaving protease. Blood. 2001; 98:1654-1661).

Mutations in the ADAMTS13 gene have been shown to cause TTP (Levy G G,Nichols W C, Lian E C, Foroud T, McClintick J N, McGee B M, Yang A Y,Siemieniak D R, Stark K R, Gruppo R, Sarode R, Shurin S B,Chandrasekaran V, Stabler S P, Sabio H, Bouhassira E E, Upshaw J D, Jr.,Ginsburg D, Tsai H M. Mutations in a member of the ADAMTS gene familycause thrombotic thrombocytopenic purpura. Nature. 2001; 413:488-494).Idiopathic TTP, often caused by autoantibodies inhibiting ADAMTS-13activity, is a more common disorder that occurs in adults and olderchildren and can recur at regular intervals in 11% to 36% of patients(Tsai H M, Lian E C. Antibodies to von Willebrand factor-cleavingprotease in acute thrombotic thrombocytopenic purpura. N Engl J Med.1998; 339:1585-1594; Furlan M, Lammle B. Deficiency of von Willebrandfactor-cleaving protease in familial and acquired thromboticthrombocytopenic purpura. Baillieres Clin Haematol. 1998; 11:509-514).

Non neutralizing autoantibodies could also inhibit ADAMTS13 activity byinducing clearance from circulation (Scheiflinger F, Knobl P, TrattnerB, Plaimauer B, Mohr G, Dockal M, Dorner F, Rieger M. NonneutralizingIgM and IgG antibodies to von Willebrand factor-cleaving protease(ADAMTS-13) in a patient with thrombotic thrombocytopenic purpura.Blood. 2003; 102:3241-3243). Plasma ADAMTS13 activity in healthy adultsranges from 50% to 178% (Moake J L. Thrombotic thrombocytopenic purpuraand the hemolytic uremic syndrome. Arch Pathol Lab Med. 2002;126:1430-1433). In most patients with familial or acquired TTP, plasmaADAMTS13 activity is absent or less than 5% of normal activity. Withouttreatment, the mortality rate for TTP exceeds 90%, but plasma therapyhas reduced mortality to about 20% (Moake J L. Thromboticthrombocytopenic purpura and the hemolytic uremic syndrome. Arch PatholLab Med. 2002; 126:1430-1433).

vWF synthesized in megakaryocytes and endothelial cells is stored inplatelet α-granules and Weibel-Palade bodies, respectively, as ultralarge vWF (UL-vWF) (Moake J L, Rudy C K, Troll J H, Weinstein M J,Colannino N M, Azocar J, Seder R H, Hong S L, Deykin D. Unusually largeplasma factor VIII:von Willebrand factor multimers in chronic relapsingthrombotic thrombocytopenic purpura. N Engl J Med. 1982; 307:1432-1435;Wagner D D, Olmsted J B, Marder V J. Immunolocalization of vonWillebrand protein in Weibel-Palade bodies of human endothelial cells. JCell Biol. 1982; 95:355-360; Wagner D D, Bonfanti R. von Willebrandfactor and the endothelium. Mayo Clin Proc. 1991; 66:621-627; Sporn L A,Marder V J, Wagner D D. von Willebrand factor released fromWeibel-Palade bodies binds more avidly to extracellular matrix than thatsecreted constitutively. Blood. 1987; 69:1531-1534; Tsai H M, Nagel R L,Hatcher V B, Sussman, I I. Endothelial cell-derived high molecularweight von Willebrand factor is converted into the plasma multimerpattern by granulocyte proteases. Biochem Biophys Res Commun. 1989;158:980-985; Tsai H M, Nagel R L, Hatcher V B, Sussman, I I. Multimericcomposition of endothelial cell-derived von Willebrand factor. Blood.1989; 73:2074-2076). Once secreted from endothelial cells, these UL-vWFmultimers are cleaved by ADAMTS13 in circulation into a series ofsmaller multimers at specific cleavage sites within the vWF molecule(Tsai H M, Nagel R L, Hatcher V B, Sussman, I I. Endothelialcell-derived high molecular weight von Willebrand factor is convertedinto the plasma multimer pattern by granulocyte proteases. BiochemBiophys Res Commun. 1989; 158:980-985; Dent J A, Galbusera M, Ruggeri ZM. Heterogeneity of plasma von Willebrand factor multimers resultingfrom proteolysis of the constituent subunit. J Clin Invest. 1991;88:774-782; Furlan M, Robles R, Affolter D, Meyer D, Baillod P, LammleB. Triplet structure of von Willebrand factor reflects proteolyticdegradation of high molecular weight multimers. Proc Natl Acad Sci USA.1993; 90:7503-7507).

ADAMTS13 cleaves at the Tyr842-Met843 bond in the central A2 domain ofthe mature vWF subunit and requires zinc or calcium for activity (Dent JA, Berkowitz S D, Ware J, Kasper C K, Ruggeri Z M. Identification of acleavage site directing the immunochemical detection of molecularabnormalities in type IIA von Willebrand factor. Proc Natl Acad Sci USA.1990; 87:6306-6310). vWF exists in “ball-of-yarn” and filamentous formas seen by electron microscopy (Slayter H, Loscalzo J, Bockenstedt P,Handin R I. Native conformation of human von Willebrand protein.Analysis by electron microscopy and quasi-elastic light scattering. JBiol. Chem. 1985; 260:8559-8563). Furthermore, atomic force microscopyconfirms that vWF exits in a globular conformation under staticconditions and an unfolded filamentous state after exposure to shearstress (Siedlecki C A, Lestini B J, Kottke-Marchant K K, Eppell S J,Wilson D L, Marchant R E. Shear-dependent changes in thethree-dimensional structure of human von Willebrand factor. Blood. 1996;88:2939-2950). This could occur also in vivo when one end of the vWFfilament is anchored to a surface.

Thrombi of TTP patients consist of little fibrin and mainly of vWF andplatelets, suggesting vWF-mediated platelet aggregation as a cause ofthrombosis (Asada Y, Sumiyoshi A, Hayashi T, Suzumiya J, Kaketani K.Immunohistochemistry of vascular lesion in thrombotic thrombocytopenicpurpura, with special reference to factor VIII related antigen. ThrombRes. 1985; 38:469-479). Patients with relapsing TTP have ultra-largemultimers in the plasma. The UL-vWF multimers accumulate over timebecause the persistence of the inhibitor (Anti-ADAMTS13 Ab) decreasesADAMTS13 activity. The UL-vWF multimers are hyperactive and unfold as aresult of shear stress causing platelet aggregation, resulting inintravascular thrombosis (Tsai H M. Von Willebrand factor, ADAMTS13, andthrombotic thrombocytopenic purpura. J Mol Med. 2002; 80:639-647; Tsai HM. Deficiency of ADAMTS-13 in thrombotic and thrombocytopenic purpura. JThromb Haemost. 2003; 1:2038-2040; discussion 2040-2035).

It is believed that the presence of hyper-reactive UL-vWF multimers inthe plasma due to ADAMTS13 deficiency could be associated with anincreased risk of arterial thrombosis linked to coronary heart disease.

As ADAMTS proteins have been implicated in a number of diseases andconditions, there is a need in the art for methods of large scaleproduction of recombinant ADAMTS proteins having high specificactivities, which are suitable for pharmaceutical formulation andadministration. The present invention provides methods which satisfythese and other needs in the art for the production and purification ofADAMTS proteins.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods of expressing adisintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)protein. In certain embodiments, the methods provided hereinadvantageously result in increased expression levels and in theproduction of ADAMTS proteins with substantially improved activities.These improved properties may be achieved, in one example, bysupplementing the culture medium used for the expression of ADAMTSproteins with increased levels of various components, such as calcium,zinc, or nicotinamide (vitamin B3). In a preferred embodiment, theADAMTS protein is an ADAMTS13 protein.

In another aspect, the invention relates to methods of increasing theexpression and/or the activity levels of an ADAMTS protein by culturingrecombinant cells harboring a nucleic acid encoding an ADAMTS protein inanimal protein-free and chemically defined mediums under batch,fed-batch or continuous cell-culture conditions. In some embodiments,these methods comprise the use of batch, fed-batch, perfusion, orchemostat cell-cultivation that may be performed in either cellsuspension or cell adherent fashions. In a preferred embodiment, theADAMTS protein is an ADAMTS13 protein.

In another aspect, the present invention provides methods of reducingthe amount of activity lost during the purification of an ADAMTSprotein. In certain embodiments, the methods comprise supplementingpurification buffers with additional levels of calcium and/or zinc. In aspecific embodiment, the methods relate to stabilizing the activity ofan ADAMTS protein during ultrafiltration and/or diafiltration steps usedduring purification. In a preferred embodiment, the ADAMTS protein is anADAMTS13 protein.

In yet another aspect, the present invention relates to methods ofproducing an ADAMTS protein with increased activity for use in thepreparation of a pharmaceutical composition. In certain embodiments,these methods comprise the use of animal protein-free and/or chemicallydefined culture mediums under conditions suitable for increasedexpression of an ADAMTS protein having increased activities. In apreferred embodiment, the ADAMTS protein is an ADAMTS13 protein.

In a related aspect, the present invention provides animal protein-free,oligopeptide-free, and chemically defined mediums that are useful forthe expression of ADAMTS proteins having high specific activities. In apreferred embodiment, the ADAMTS protein is an ADAMTS13 protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B. Volumetric FRETS-vWF73 productivity of recombinanthuman ADAMTS13 expressed in CHO cells cultured in animal protein-freemedium under varying temperature and pH conditions. The results of thevarious cell-culture experiments are shown as (A) contour plot and (B)surface plot representations of normalized volumetric ADAMTS13FRETS-VWF73 productivity as a function of culture temperature and pH.

FIG. 2A and FIG. 2B. Specific activity levels (FRETS-VWF73/antigen byELISA) of CHO cells expressing recombinant human ADAMTS13 cultured inanimal protein-free medium under varying temperature and pH conditions.The results of the various cell-culture experiments are shown as (A)contour plot and (B) surface plot representations of specific activityas a function of culture temperature and pH.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The ADAMTS proteins (i.e., ADAMTS-1 to ADAMTS-20) are a family ofsecreted zinc metalloproteinases that share a common modular domainorganization (for review, see, Flannery C. R., Front Biosci. 2006 Jan1;11:544-69). All of the ADAMTS protein share a common core domainarchitecture, consisting of a signal peptide, followed by a prodomain, azinc-dependent metalloproteinase catalytic domain, a disintegrin-likedomain, a thrombospondin type I repeat, a cysteine-rich domain, and aspacer domain (Apte S.S., J Biol Chem. 2009 Nov. 13; 284(46):31493-7).Additionally, all but ADAMTS-4 contain at least one more thrombospondintype I repeat domain, and many of the ADAMTS protein contain one or moreadditional ancillary domains. Notably, it has been reported that allADAMTS protein appear to contain at least one calcium binding site andat least one zinc binding site located within the metalloproteinasecatalytic domain (Andreini et al., J. Proteome Res., 2005,4 (3), pp881-888).

Biological roles for ADAMTS proteins have been reported for variousdiseases and conditions, including, Antiangiogenesis, Renal interstitialfibrosis, Bone remodeling, Ovarian folliculogenesis, Atherosclerosis,Urogenital development, and Tumor growth/remodeling (ADAMTS-1);Ehler-Danlos syndrome type 7C and Bovine dermatopraxis (ADAMTS-2);Arthritis, Atherosclerosis, and Tendinopathy (ADAMTS-4); Arthritis andGlioblastoma (ADAMTS-5); Arthritis (ADAMTS-7); Antiangiogenesis, Brainmalignancy, Arthritis, and Atherosclerosis (ADAMTS-8); Arthritis(ADAMTS-9, -12); Thrombotic thrombocytopenic purpura (ADAMTS-13); andAntithrombosis/stroke (ADAMTS18) (for review, see, Lin and Liu, OpenAccess Rheumatology Research and Reviews 2009:1 121-131).

Recombinant ADAMTS13 (A13) has been expressed before in mammalian cells,however the specific activity varies widely dependent on the cellculture conditions. It has been found that many commercially availableculture mediums are not sufficient for expression of A13 with highspecific activities, expressed as the ratio of activity, measured byFRETS-VWF73 assay, to antigen content, as determined by ELISA. In oneaspect, the methods provided herein are based on several advantageousfindings that allow for cell-culture expression of A13 having increasedlevels of total and specific activity.

The studies described herein demonstrate that a certain minimumconcentration of calcium in the culture medium, about 0.5 mM to about1.5 mM, is required for expression of active A13. Additionally, it wasfound that by supplementing the culture medium with increased levels ofzinc, the resultant expressed A13 protein had higher total and specificactivities. For example, in cultures supplemented with additional zincat 2 to 3 times the normal concentration, as compared to concentrationsfound in standard chemically defined mediums such as DMEM/F12 basedmediums, the specific activity of A13 was significantly increased.Furthermore, an additional increase in the specific activity of A13could be achieved by increasing the nicotinamide (vitamin B3)concentration from about 2 mg/L, as in standard DMEM/F12 based mediums,to about 7 mg/L. By culturing recombinant CHO or HEK293 cells in mediumssupplemented with additional calcium, zinc, and/or nicotinamide,specific activities of at least about 500 mU/μg, 1000 mU/μg, up to about2000 mU/μg could be achieved in large scale expression cultures ofrecombinant human A13. These levels of specific activity forrecombinantly produced A13 proteins have not been reported previously.Advantageously, these increased levels of Al3 activity were achieved inchemically defined medium free of animal derived components, allowingfor consistent and reproducible expression of ADAMTS proteins on a largescale suitable for production of proteins for pharmaceuticalformulation. Furthermore, these mediums are even free of recombinantproteins, e.g. insulin, resulting in products that can more safely beused in formulations for pharmaceutical administration.

The present disclosure also provides methods that prevent the loss ofactivity and specific activity during standard ultra/diafiltration andother purification steps. Advantageously, it was found that bysupplementing the buffer used for diafiltration with additional calciumand zinc, a significant reduction in the loss of A13 activity, asmeasured by a FRETS-VWF73 assay, could be achieved.

Accordingly, due to the shared structure-function relationship betweenthe ADAMTS family of secreted metalloproteinases, the methods providedby the present invention allow for the expression of all ADAMTS proteinsin cell culture and recovery from the cell medium.

II. DEFINITIONS

As used herein, the terms “vitamin B3,” “nicotinamide,” “niacinamide,”“niacin,” and “nicotinic acid” may be used interchangeably to refer toany member of the B3 family of vitamins. Accordingly, any member of thisfamily may be used to supplement medium used in the methods of thepresent invention.

As used herein, an “ADAMTS protein” refers to a polypeptide of thedisintegrin and metalloproteinase with thrombospondin type I motifsfamily of metalloproteinases. Members of this family include the humanproteins ADAMTS1 (NM_006988), ADAMTS2 (NM_014244; NM_021599), ADAMTS3(NM_014243), ADAMTS4 (NM_005099), ADAMTS5 (NM_007038), ADAMTS6(NM_014273), ADAMTS7 (NM_0142727), ADAMTS8 (NM_007037), ADAMTS9(NM_182920; NM_182921; NM_020249), ADAMTS10 (NM_030957), ADAMTS12(NM_030955), ADAMTS13 (NM_139025; NM_139026; NM_139027; NM_139028),ADAMTS14 (NM_139155; NM_080722), ADAMTS15 (NM_139055), ADAMTS16(NM_139056), ADAMTS17 (NM_139057), ADAMTS18 (NM_199355; NM_139054),ADAMTS19 (NM_133638), and ADAMTS20 (NM_025003, NM_175851). ADAMTSproteins include both full-length proteins and partial polypeptides thatdisplay at least partial biological activity, for example, at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the activitydemonstrated by the full-length protein, in particular the proteaseactivity demonstrated by the full length protein. In certain instances,an ADAMTS protein will be post-translationally modified either in vivoor in vitro, for example, by enzymatic or chemical means. It isunderstood that the ADAMTS proteins of the present invention includealternatively spliced isoforms, conservatively modified proteins,substantially identical proteins, homologues, and the like.

In the context of the present invention, an ADAMTS protein embraces anymember of the ADAMTS family from, for example, a mammal such as aprimate, human, monkey, rabbit, pig, rodent, mouse, rat, hamster,gerbil, canine, feline, and biologically active derivatives thereof.Mutant and variant ADAMTS proteins having activity are also embraced, asare functional fragments and fusion proteins of the ADAMTS proteins.Furthermore, the ADAMTS proteins of the invention may further comprisetags that facilitate purification, detection, or both. The ADAMTSproteins described herein may further be modified with a therapeuticmoiety or a moiety suitable imaging in vitro or in vivo.

As used herein, an “ADAMTS13 protein” refers to any protein orpolypeptide with ADAMTS13 activity, particularly the ability to cleavethe peptide bond between residues Tyr-842 and Met-843 of VWF. In anexemplary embodiment, an ADAMTS13 protein refers to a polypeptidecomprising an amino acid sequence that is highly similar to that ofNP_620594 (ADAMTS13 isoform 1, preproprotein) or amino acids 75 to 1427of NP_620594 (ADAMTS13 isoform 1, mature polypeptide). In anotherembodiment, an ADAMTS13 protein refers to a polypeptide comprising anamino acid sequence that is highly similar to that of NP_620596(ADAMTS13 isoform 2, preproprotein) or amino acids 75 to 1371 ofNP_620594 (ADAMTS13 isoform 2, mature polypeptide). In yet anotherembodiment, ADAMTS13 proteins include polypeptides comprising an aminoacid sequence highly similar to that of NP_620595 (ADAMTS13 isoform 3,preproprotein) or amino acids 75 to 1340 of NP_620595 (ADAMTS13 isoform1, mature polypeptide). As used herein, an ADAMTS13 protein includesnatural variants with vWF cleaving activity and artificial constructswith vWF cleaving activity. As used in the present invention, ADAMTS13encompasses any natural variants, alternative sequences, isoforms ormutant proteins that retain some basal activity. Examples of ADAMTS13mutations found in the human population include, without limitation,R7W, V88M, H96D, R102C, R193W, T196I, H234Q, A250V, R268P, W390C, R398H,Q448E, Q456H, P457L, C508Y, R528G, P618A, R625H, I673F, R692C, A732V,S903L, C908Y, C951G, G982R, C1024G, A1033T, R1095W, R1123C, C1213Y,T12261, G1239V, R1336W, many of which have been found associated withthrombotic thrombocytopenic purpura (TTP). ADAMTS13 proteins alsoincludes polypeptides containing post-translational modifications. Forexample, ADAMTS13 has been shown to be modified by N-acetylglucosamine(GlcNAc) at residues 614, 667, and 1354, and it has been predicted thatresidues 142, 146, 552, 579, 707, 828, and 1235 may also be modified inthis fashion.

Proteolytically active recombinant ADAMTS13 may be prepared byexpression in mammalian cell cultures, as described in Plaimauer et al.,(2002, Blood. 15;100(10):3626-32) and US 2005/0266528, the disclosuresof which are herein incorporated by reference in their entireties forall purposes. Methods of recombinant culture of ADAMTS13 expressingcells are disclosed in Plaimauer B, Scheiflinger F. (Semin Hematol. 2004January; 41(1):24-33, the disclosure of which is herein incorporated byreference in its entirety for all purposes).

As used herein, “one unit of ADAMTS activity” is defined as the amountof activity in 1 mL of pooled normal human plasma, regardless of theassay being used. For example, when the ADAMTS protein is ADAMTS13, oneunit of ADAMTS13 FRETS-VWF73 activity is the amount of activity neededto cleave the same amount of FRETS-VWF73 substrate (Kokame et al., Br JHaematol. 2005 April; 129(1):93-100) as is cleaved by one mL of poolednormal human plasma. Conveniently, ADAMTS13 activity may be determinedby functional assays, such as functional assays employing modified vonWillebrand factor peptides as substrate for ADAMTS13 (Tripodi et al. JThromb Haemost. 2008 September;6(9): 1534-41). A preferred method ofdetermining recombinant human ADAMTS13 activity is disclosed inGerritsen et al. (Assay of von Willebrand factor (vWF)-cleaving proteasebased on decreased collagen binding affinity of degraded vWF: a tool forthe diagnosis of thrombotic thrombocytopenic purpura (TTP). ThrombHaemost 1999; 82: 1386-1389). In one embodiment, to be considered as aADAMTS13 protein as defined above, a polypeptide or protein must have atleast 1% of the vWF cleaving activity of native ADAMTS13. In otherembodiments, an ADAMTS13 protein will contain at least 10% of theactivity of native ADAMTS13. In yet other embodiments, an ADAMTS13protein will contain at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or 100% of the activity of native ADAMTS13. The quantity of anADAMTS13 protein may also be determined by measurement of an ADAMTS13antigen, for example using the ELISA method disclosed in Rieger et al.,(2006, Thromb Haemost. 2006 95(2):212- 20).

As used herein, “μg of ADAMTS13” or “μg of ADAMTS13 antigen” means anamount of ADAMTS13 which provides the same level of detectable proteinby ELISA assay as 1 mL of pooled normal human plasma. This is based onpublished estimates of 1 mg of ADAMTS13 being present in 1 mL of normalhuman plasma, and is thus an approximate measurement.

As used herein, the term “biologically active derivative”, when used inthe context of an ADAMTS protein, also embraces polypeptides obtainedvia recombinant DNA technology. This may include any method known in theart for (i) the production of recombinant DNA by genetic engineering,e.g., via reverse transcription of RNA and/or amplification of DNA, (ii)introducing

Attorney Docket No.: 008073-5020-US05 recombinant DNA into prokaryoticor eukaryotic cells by transfection, i.e., via electroporation ormicroinjection, (iii) cultivating said transformed cells, e.g., in acontinuous or batch-wise manner, (iv) expressing an ADAMTS protein,e.g., constitutively or upon induction, and (v) isolating said ADAMTSprotein, e.g., from the culture medium or by harvesting the transformedcells, in order to (vi) obtain substantially purified recombinant ADAMTSprotein, e.g., via ion exchange chromatography, size exclusionchromatography, affinity chromatography, hydrophobic interactionchromatography, and the like. The term “biologically active derivative”includes also chimeric molecules such as e.g. an ADAMTS protein, orfunctional fragment thereof, in combination with a second polypeptide,e.g., an immunoglobulin Fc domain or an albumin domain, in order toimprove the biological/pharmacological properties such as e.g., halflife of the ADAMTS protein in the circulation system of a mammal,particularly a human.

As used herein, the term “ultrafiltration” encompasses a variety ofmembrane filtration methods in which hydrostatic pressure forces aliquid against a semi-permeable membrane. Suspended solids and solutesof high molecular weight are retained, while water and low molecularweight solutes pass through the membrane. This separation process isoften used for purifying and concentrating macromolecular (10³-10⁶ Da)solutions, especially protein solutions. A number of ultrafiltrationmembranes are available depending on the size of the molecules theyretain. Ultrafiltration is typically characterized by a membrane poresize between 2 nm and 0.05 μm and operating pressures between 1 and 10bar, and is particularly useful for separating colloids like proteinsfrom small molecules like sugars and salts. In contrast, nanofiltrationis another pressure-driven filtration method typically characterized bya membrane pore size between 0.5 and 2 nm and operating pressuresbetween 5 and 40 bar. Nanofiltration is frequently used to achieve aseparation between sugars, other organic molecules and multivalent saltson one hand and monovalent salts and water on the other. Generally,ultrafiltration may be performed in either dead-end filtration mode ortangential flow filtration (TFF) mode.

As used herein, the term “diafiltration” refers to another membranefiltration method, sometimes referred to as tangential flow filtration(TFF), wherein the liquid is pumped tangentially along the surface of anultrafiltration membrane. Typically, the retentate liquid is dilutedwith diafiltration buffer after passing over the membrane andsubsequently returned to the membrane in a continuous flow process.Generally, diafiltration may be performed in either dead-end filtrationmode or tangential flow filtration (TFF) mode. As such, a single systemmay be used for both ultrafiltration and diafiltration operations, forexample, to first concentrate the sample using ultrafiltration and thenperform buffer exchange by diafiltration.

As used herein, the term “polyamine” refers to any of a group of organiccompounds composed of carbon, nitrogen, and hydrogen, and containing twoor more amino groups. For example, the term encompasses moleculesselected from the group consisting of cadaverine, putrescine, agmatine,ornithine, spermine and spermidine.

In certain embodiments of the chemically defined culture medium used forthe expression of an ADAMTS protein, the concentration of the polyamineis present in a concentration ranging from about 0.5 mg/L to about 30mg/L, or from about 0.5 mg/L to about 20 mg/L, or from about 0.5 mg/L toabout 10 mg/L, or from about lmg/L to about 10 mg/L, or from about 2mg/L to about 10 mg/L, or from about 2 mg/L to about 8 mg/L, or fromabout 2 mg/L to about 5 mg/L in the medium. In one specific embodiment,the polyamine is putrescine at a concentration from about 2 mg/L toabout 8 mg/L.

As used herein, the term “chemically defined medium” refers to asynthetic growth medium in which the identity and concentration of allthe components are known. Chemically defined mediums do not containbacterial, yeast, animal, or plant extracts, although they may or maynot include individual plant or animal-derived components (e.g.,proteins, polypeptides, etc.). Non-limiting examples of commerciallyavailable chemically defined mediums include, various EX-CELL® mediums(SAFC Biosciences, Inc), various Dulbecco's Modified Eagle's (DME)mediums (Sigma-Aldrich Co; SAFC Biosciences, Inc), Ham's NutrientMixture (Sigma-Aldrich Co; SAFC Biosciences, Inc), and the like. Methodsof preparing chemically defined culture mediums are known in the art,for example in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007/077217,and U.S. Patent Application Publication Numbers 2008/0009040 and2007/0212770, the disclosures of which are incorporated herein byreference in their entireties for all purposes.

As used herein, the term “oligopeptide-free culture medium” refers to aprotein-free medium that does not comprise oligopeptides, such as, e.g.,oligopeptides derived from a protein hydrolysate. In one embodiment, themedium does not comprise oligopeptides having twenty or more aminoacids. In one embodiment of the present invention, the medium does notcomprise oligopeptides having fifteen or more amino acids. In anotherembodiment of the invention, the medium does not comprise oligopeptideshaving ten or more amino acids. In one embodiment the medium does notcomprise oligopeptides having seven or more amino acids. In anotherembodiment the medium does not comprise oligopeptides having five ormore amino acids. In still another embodiment the medium does notcomprise oligopeptides having three or more amino acids. According to afurther embodiment of the present invention, the medium does notcomprise oligopeptides having two or more amino acids. Methods ofpreparing oligopeptide-free culture medium are known in the art, forexample in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007/077217, andU.S. Patent Application Publication Numbers 2008/0009040 and2007/0212770, the disclosures of which are incorporated herein byreference in their entireties for all purposes.

As used herein, the term “serum-free culture medium” refers to a culturemedium that is not supplemented with an animal serum. Althoughoftentimes serum-free mediums are chemically defined mediums, serum-freemediums may be supplemented with discrete animal or plant proteins orprotein fractions. Methods of preparing serum-free culture medium areknown in the art, for example in U.S. Pat. Nos. 6,171,825 and 6,936,441,WO 2007/077217, and U.S. Patent Application Publication Numbers2008/0009040 and 2007/0212770, the disclosures of which are incorporatedherein by reference in their entireties for all purposes.

As used herein, the term “animal protein-free culture medium” refers toa culture medium that is not supplemented with an animal serum, protein,or protein fraction. Although oftentimes animal protein-free culturemediums are chemically defined mediums, animal protein-free culturemediums may contain plant or yeast hydrolysates. Methods of preparinganimal protein-free culture medium are known in the art, for example inU.S. Patent Numbers 6,171,825 and 6,936,441, WO 2007/077217, and U.S.Patent Application Publication Numbers 2008/0009040 and 2007/0212770,the disclosures of which are incorporated herein by reference in theirentireties for all purposes.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

As used herein, the term “about” denotes an approximate range of plus orminus 10% from a specified value. For instance, the language “about 20%”encompasses a range of 18-22%.

III. ADAMTS PROTEIN EXPRESSION

In one aspect, the present invention provides methods of expressing adisintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)protein having high specific activity. In one embodiment, the methodcomprises culturing a cell harboring a nucleic acid encoding an ADAMTSprotein in culture medium supplemented with at least one componentselected from calcium, zinc, and nicotinamide (Vitamin B3). In aspecific embodiment, an ADAMTS protein is expressed in a mediumsupplemented with at least two components selected from calcium, zinc,and nicotinamide (Vitamin B3). In yet another embodiment, the culturemedium is supplemented with calcium, zinc, and nicotinamide (VitaminB3). In certain embodiments, the culture medium used for the expressionof an ADAMTS protein may comprise an animal protein-free, anoligopeptide-free, or a chemically defined medium.

In one aspect, methods are provided for the production of an ADAMTSprotein. In one embodiment, the methods comprise the steps of culturinga cell harboring a nucleic acid encoding an ADAMTS protein in a culturemedium supplemented with at least one of zinc, calcium, andnicotinamide; removing a fraction of the supernatant from the culture;performing a centrifugation or a filtration step to remove any residualcells; performing an ultrafiltration step to concentrate the ADAMTSprotein; and performing a diafiltration step with a buffer comprising atleast calcium or zinc. In some embodiments, the concentration of calciummay be at least about 0.1 mM, 0.3 mM, 0.5 mM, 0,75 mM, 1 mM, 1.5 mM, 2mM, 3 mM, 5 mM or more than 5 mM calcium. In other embodiments, theconcentration of zinc may be at least about 0.5 μM, 1 μM, 2 μM, 3 μM, 5μM, 10 μM or more than 10 μM zinc. In a certain embodiment the harvest,cell free supernatant or the diafiltration buffer contains combinationsof calcium and zinc in the above mentioned concentrations. In a certainembodiment the cut off of the ultra and/or diafiltration membranes canbe e.g. about 150 kD or 125 kD or 100 kD or 75 kD or 50 kD or 30 kD or10 kD or less than 10 kD. In a certain embodiment, the ADAMTS protein isADAMTS13 or a biologically active derivative thereof. In a specificembodiment, the ADAMTS13 protein is a human ADAMTS13 protein orbiologically active derivative thereof. In certain embodiments, theculture medium used in the method may be an animal protein-free,oligopeptide-free, or chemically defined medium.

In one embodiment, the method further comprises a purification stepselected from the group consisting of ion exchange chromatography, sizeexclusion chromatography, affinity chromatography, and hydrophobicinteraction chromatography.

In yet another embodiment, zinc supplementation may be provided byaddition of a protein or polypeptide preparation containing zinc. Forexample, typical preparations of insulin contain zinc at concentrationssuch that medium supplementation with between about 1 mg/L and about 10mg/L insulin would also result in supplementation of about 0.03 μM toabout 1.5 μM zinc, as calculated from the detailed monograph for NovolinN InnoLet SubQ, recombinant human insulin, which can be found on theMedscape server. Accordingly, in one embodiment, culture medium used forthe expression of an ADAMTS protein may be supplemented with azinc-containing insulin preparation.

The basal medium, which is supplemented with zinc, calcium, and/ornicotinamide (vitamin B3) as disclosed herein, chosen for culturing thehost cell line is not critical to the present invention and may be anyone of, or combination of, those known to the art which are suitable forculturing mammalian cells. Medium such as Dulbecco's Modified EagleMedium, Ham's F-12 Medium, Eagle's Minimal Essential Medium andRPMI-1640 Medium and the like are commercially available. The additionof growth factors such as recombinant insulin is optional.

In one embodiment, the basal medium used to culture a cell expressing anADAMTS protein (e.g., ADAMTS13) may comprise a mixture of one or morecommercially available chemically defined mediums, for example,Dulbecco's Modified Eagle Medium (DMEM) and Ham's F-12 Medium, which hasbeen supplemented with one or more components other than zinc, calcium,and nicotinamide (vitamin B3). Non-limiting examples of components thatmay be used to supplement a commercially available medium include,essential amino acids (e.g., glutamine), non-ionic surfactants (e.g.,Synperonic), primary amines (e.g., ethanolamine), polyamines (e.g.,putrescine), trace metals (e.g., iron), and buffering agents (e.g.,sodium bicarbonate). In a specific embodiment, the medium is a BCSmedium as provided in Table 1. In another specific embodiment, themedium is a BACD medium, for example, BACD-A13 medium.

TABLE 1 Composition of cell culture medium BCS. Component Concentration[g/kg] DMEM/HAM'S F12 11.75 L-Glutamine 0.9 Synperonic 1.00 Ethanolamine0.00153 Putrescine•2HCl 0.0036 FeSO4•7H2O 0.0006 NaHCO3 2.0

Historically, animal cells have been cultured in media containing animalserum. However, such media are incompletely defined and carry the riskof infection. Those in the art have therefore devised “protein-free”media that are either completely free of any protein or at least arefree of any protein that is not recombinantly produced. Human serumalbumin is commonly used as a serum-free culture supplement for theproduction of recombinant proteins. Preferred media include thosedisclosed in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007/077217, andU.S. Patent Application Publication Numbers 2008/0009040 and2007/0212770.

Optionally, a non- ionic surface-active agent such as a polypropyleneglycol (e.g. Pluronic° F-61 , Pluronic° F-68, Pluronic° F-71, Pluronic°F-108 or a Synperonic®) maybe added to the medium as a defoaming agent.This agent is generally applied to protect the cells from the negativeeffects of aeration (“sparging”). The amount of non-ionic surface-activeagent may range between 0.05 and 10 g/L, preferably between 0.1 and 5g/L.

The medium of U.S. Pat. No. 6 936 441 is particularly well suited to theculturing of CHO cells but may be used with other cells as well. Afurther suitable medium is the oligopeptide -free medium disclosed inU.S. Patent Application No. 2007/0212770 (Baxter International Inc.,Baxter Healthcare S.A.).

TABLE 2 Exemplary concentrations of zinc, calcium, and nicotinamide(vitamin B3) concentration that can be used to supplement culturemediums useful for the expression of an ADAMTS protein. At least 2 μMZinc Var. 1 At least 0.5 mM calcium Var. 2 At least 2 mg/L Var. 3nicotinamide At least 5 μM Zinc Var. 4 At least 1.5 mM calcium Var. 5 Atleast 7 mg/L Var. 6 nicotinamide Between 2 and 12 μM Var. 7 Between 0.5and 1.5 mM Var. 8 Between 2 and 7 mg/L Var. 9 zinc calcium nicotinamideBetween 5 μM and Var. At least 0.6 mM, 0.7 mM, Var. At least 3 mg/L, 4mg/L, Var. 12 μM zinc 10 0.8 mM, 0.9 mM, 1.0 mM, 11 5 mg/L, 6 mg/L, 121.1 mM, 1.2 mM, 1.3 mM, 7 mg/L, 8 mg/L, 1.4 mM, 1.5 mM, 1.6 mM, 9 mg/L,10 mg/L, 1.7 mM, 1.8 mM, 1.9 mM, 15 mg/L, 20 mg/L, 2.0 mM, 2.25 mM, 2.5mM, or more 2.75 mM, 3.0 mM, 3.5 mM, nicotinamide 4.0 mM, 4.5 mM, 5.0mM, or more calcium At least 3 μM, 4 μM, Var. 5 μM, 6 μM, 7 μM, 13 8 μM,9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, ormore zinc *Var. = Variation

In one embodiment, the present invention provides a method forexpressing a disintegrin and metalloproteinase with thrombospondinmotifs (ADAMTS) protein, the method comprising culturing a cellharboring a nucleic acid encoding an ADAMTS protein in a culture mediumcomprising at least one of zinc at a concentration of at least about 2μM or calcium at a concentration of at least about 0.5 mM. In oneembodiment, the ADAMTS protein is ADAMTS1. In another embodiment, theADAMTS protein is ADAMTS2. In another embodiment, the ADAMTS protein isADAMTS3. In another embodiment, the ADAMTS protein is ADAMTS4. Inanother embodiment, the ADAMTS protein is ADAMTS5. In anotherembodiment, the ADAMTS protein is ADAMTS6. In another embodiment, theADAMTS protein is ADAMTS7. In another embodiment, the ADAMTS protein isADAMTS8. In another embodiment, the ADAMTS protein is ADAMTS9. Inanother embodiment, the ADAMTS protein is ADAMTS10. In anotherembodiment, the ADAMTS protein is ADAMTS11. In another embodiment, theADAMTS protein is ADAMTS12. In another embodiment, the ADAMTS protein isADAMTS13. In another embodiment, the ADAMTS protein is ADAMTS14. Inanother embodiment, the ADAMTS protein is ADAMTS15. In anotherembodiment, the ADAMTS protein is ADAMTS16. In another embodiment, theADAMTS protein is ADAMTS17. In another embodiment, the ADAMTS protein isADAMTS18. In another embodiment, the ADAMTS protein is ADAMTS19. Inanother embodiment, the ADAMTS protein is ADAMTS20. In a preferredembodiment, the ADAMTS protein is ADAMTS 13 .

In one embodiment, the culture medium contains at least about 2 μM zinc.In another embodiment, the culture medium contains between about 2 μM toabout 12 μM zinc. In yet another embodiment, the culture medium containsat least about 5 μM zinc. In one embodiment, the culture medium containsbetween about 5 μM to about 12 μM zinc. In another embodiment, theculture medium contains at least about 0.5 mM calcium. In yet anotherembodiment, the culture medium contains between about 0.5 mM and about1.5 mM calcium. In one embodiment, the culture medium contains at leastabout 2 μM zinc and at least about 0.5 mM calcium.

In yet other embodiments, it has been found that the addition ofnicotinamide (vitamin B3) further enhances the expression and specificactivity of ADAMTS proteins in cell culture. In one embodiment, theculture medium further comprises at least about 2 mg/L nicotinamide(vitamin B3). In another embodiment, the culture medium furthercomprises at least about 7 mg/L nicotinamide (vitamin B3). In yetanother embodiment, the culture medium contains between about 2 mg/L andabout 10 mg/L nicotinamide (vitamin B3).

In certain embodiments, the culture medium is an animal protein freeculture medium. In another embodiment, the culture medium is achemically defined medium. In certain embodiments, the culture mediummay comprise one or more polyamines. In a particular embodiment, thepolyamine is putrescine, for example, at a concentration of at least 0.5mg/L. In a specific embodiment, the culture medium contains betweenabout 2 mg/L and about 8 mg/L putrescine.

In certain embodiments, the cell or cell line used in the culture is abacterial cell, a yeast cell, an insect cell, an avian cell, or amammalian cell. In a specific embodiment, the cell line is a human cellline, a hamster cell line, or a murine cell line. In a more specificembodiment, the cell line is a CHO, BHK, or HEK cell line. In apreferred embodiment, the cell line is a CHO cell line.

In certain embodiments, the nucleic acid encoding the ADAMTS proteincomprises a control sequence operably linked to the nucleotide sequenceencoding the ADAMTS protein. In one embodiment, the control sequence isa promoter. In certain embodiments, the promoter is a constitutivepromoter. In other embodiments, the promoter is an inducible promoter.

In certain embodiments, the methods provided herein comprise the use ofa continuous cell culture system (i.e., continuous cell-cultivation). Inone embodiment, the continuous culture system is a chemostat culturesystem (i.e., chemostat cell-cultivation). In another embodiment, thecontinuous culture system is a turbidostat culture system (i.e.,turbidostat cell-cultivation). In yet another embodiment, the continuousculture system is a perfusion culture system (i.e., perfusioncell-cultivation). In certain embodiments, the continuous culture systemmay be operated under a suspension mode. In other embodiments, thecontinuous culture system may be operated under an adherent mode. Incertain embodiments, the adherent mode comprises the use of amicrocarrier, for example, a porous microcarrier.

In certain embodiments, the methods provided herein comprise the use ofa batch cell culture system (i.e., batch cell-cultivation). In oneembodiment, the batch culture system is a single-batch culture system(i.e., single-batch cell-cultivation). In another embodiment, the batchculture system is a fed-batch culture system (i.e., fed-batchcell-cultivation). In yet another embodiment, the batch culture systemis a repeated-batch culture system (i.e., repeated-batchcell-cultivation). In certain embodiments, the batch culture system maybe operated under a suspension mode. In other embodiments, the batchculture system may be operated under an adherent mode. In certainembodiments, the adherent mode comprises the use of a microcarrier, forexample, a porous microcarrier.

In certain embodiments, the culture will maintained at a temperaturebetween about 35° C. and about 37° C. In other embodiments, the culturewill be maintained at a pH of between about 6.9 and about 7.3. In aspecific embodiment, the culture will be maintained at a pH of betweenabout 7.05 and about 7.15.

In certain embodiments, the methods provided herein will yield ADAMTS13proteins in the culture medium (i.e., expressed ADAMTS13) with specificactivities of at least 600U per mg ADAMTS13 protein. In otherembodiments, the specific activity will be at least 800U per mg ADAMTS13protein. In other embodiments, the specific activity will be at least1000U per mg ADAMTS13 protein. In other embodiments, the specificactivity will be at least 1500U per mg ADAMTS13 protein. In otherembodiments, the specific activity will be at least 2000U per mgADAMTS13 protein.

In other embodiments, the methods provide cultures that yield at least400U of ADAMTS13 activity per L of culture per day (U/L/D). In oneembodiment, the methods provide cultures that yield at least 800U ofADAMTS13 activity per L of culture per day (U/L/D).

A. ADAMTS13

In one aspect, the present invention provides methods for expressing anADAMTS13 protein having increased total and/or specific activity.Advantageously, it was found that by supplementing a growth medium withcalcium, zinc, and/or nicotinamide (Vitamin B3), ADAMTS13 polypeptideshaving high specific activity could be recombinantly expressed andrecovered from cell culture.

Methods for the expression of ADAMTS13 are provided in WO 2009/086309,the disclosure of which is herein incorporated by reference in itsentirety for all purposes. This reference describes suitable methods andculture conditions, which may be maintained throughout the duration ofthe culture. As described herein, cell cultures used for the expressionof ADAMTS13 proteins will generally be maintained at a temperature at orabout between 34° C. and 37° C. and a pH at or about between 6.8 and7.3.

Ways of monitoring culture temperature and pH are well known in this artand generally rely on probes that are inserted into the bioreactor, orincluded in loops through which the culture medium is circulated, orinserted into extracted samples of culture medium. Suitable in-line pHsensors include the Mettler Toledo InPro 3100/125/Pt100 sensor(Mettler-Toledo Ingold, Inc., Bedford, Mass.). Ways of altering thespecified parameter in order to keep it at the predefined level are alsowell known. For example, keeping the temperature constant usuallyinvolves heating or cooling the bioreactor or the feed medium (if it isa fed-batch or continuous process); keeping the pH constant usuallyinvolves choosing and supplying enough of an appropriate buffer(typically bicarbonate) and adding acid, such as hydrochloric acid, oralkali, such as sodium hydroxide, sodium bicarbonate or a mixturethereof, to the feed medium as necessary. It is possible that thecalibration of an in-line pH probe may drift over time, such as overperiods of days or weeks, during which the cells are cultured. In thatevent, it may be beneficial to reset the in-line probe by usingmeasurements obtained from a recently calibrated off-line probe.

In one embodiment, the methods comprise culturing a cell harboring anucleic acid encoding an ADAMTS13 protein in culture medium supplementedwith at least one component selected from calcium, zinc, andnicotinamide (Vitamin B3). In a specific embodiment, an ADAMTS proteinis expressed in a medium supplemented with at least two componentsselected from calcium, zinc, and nicotinamide (Vitamin B3). In yetanother embodiment, the culture medium is supplemented with calcium,zinc, and nicotinamide (Vitamin B3). In some embodiments, the culturemedium may be an animal protein-free, an oligopeptide-free, or achemically defined culture medium.

1. Zinc Supplementation

Advantageously, it was found that increased ADAMTS13 enzymatic activityand specific activity could be recovered from a cell culture grown in amedium supplemented with zinc. For example, Example 1, demonstrates thatADAMTS13 protein expressed in culture medium containing 1.432 mg/LZnSO₄.7H₂O (5 μM zinc) has a 40% to 100% higher specific activity thanADAMTS13 protein expressed in culture medium containing only 0.432 mg/LZnSO₄.7H₂O (1.5 μM zinc) (compare, Table 10 and Table 11). Furthermore,this effect is reproducible, as shown in Example 2 (compare, Table 13and Table 14); Example 4 (Table 16 to Table 19); and Example 5 (Table 20and Table 21).

Accordingly, in one aspect, the present invention provides methods forexpressing an ADAMTS protein (e.g., ADAMTS13) having an increasedspecific activity by culturing a cell harboring a nucleic acid encodingan ADAMTS protein (e.g., ADAMTS13) in a culture medium supplemented withzinc, for example, containing at least 2 μM zinc. Similarly, the presentinvention also provides methods for preparing an ADAMTS proteincomposition (e.g., an ADAMTS13 composition) having increased totalactivity or specific activity by culturing a cell harboring a nucleicacid encoding an ADAMTS protein (e.g., ADAMTS13) in a culture mediumsupplemented with zinc, for example, containing at least 2 μM zinc.

In one embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 2 μM zinc. In another embodiment, the method comprisesculturing a cell harboring a nucleic acid encoding an ADAMTS13 proteinin a culture medium comprising at least at or about 5 μM zinc. In oneembodiment, the culture medium contains at or about between 2 μM and 12μM zinc. In another embodiment, the culture medium contains at or aboutbetween 5 μM and 12 μM zinc. In yet other embodiments, the culturemedium may contain at least at or about 2 μM, or at least at or about 3μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14μM, 15 μM, 20 μM, 25 μM, 30 μM, or more zinc. Suitable zincconcentration ranges are generally determined by cell culture toxicitiesthat may occur in the presence of high concentrations of zinc, e.g., atconcentrations greater than 20 μM, 25 μM, 30 μM, 40 μM, and the like. Aswill be understood by the skilled artisan, the extent to which zincconcentrations are inhibitory to a particular culture system will behighly dependent upon, among other factors, the type of cell used toexpress an ADAMTS protein, the components of the culture mediumutilized, and the operative mode employed for the culture (e.g., batchvs. continuous; suspension vs. adherent; chemostat vs. perfusion; etc.).In certain instances, higher zinc concentrations may be required wherecomponents of the culture medium may sequester zinc from the solution,for example, in cases where the culture medium contains albumin.Accordingly, suitable zinc concentration ranges are generally determinedby the identity of the cultured cells, medium and operative modeemployed. One of skill will readily be able to determine appropriateupper limits for the use of zinc supplementation based on the individualculture system employed.

In one embodiment, a method is provided for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein,comprising culturing a cell harboring a nucleic acid encoding anADAMTS13 protein in an animal protein and/or polypeptide free culturemedium containing at least at or about 2 μM zinc. In another embodiment,the method comprises culturing a cell harboring a nucleic acid encodingan ADAMTS13 protein in an animal protein and/or polypeptide free culturemedium comprising at least at or about 5 μM zinc. In one embodiment, theanimal protein and/or polypeptide free culture medium contains at orabout between 2 μM and 12 μM zinc. In another embodiment, the animalprotein and/or polypeptide free culture medium contains at or aboutbetween 5 μM and 12 μM zinc. In yet other embodiments, the animalprotein and/or polypeptide free culture medium may contain at least ator about 2 μM, or at least at or about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM,or more zinc. In one embodiment, the culture medium further comprises apolyamine at or about between 0.5 mg/L and 30 mg/L. In anotherembodiment, the culture medium further comprises a polyamine at or aboutbetween 2 mg/L and 8 mg/L. In a specific embodiment, the polyamine isputrescine.

In another embodiment, a method is provided for expressing a disintegrinand metalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein,comprising culturing a cell harboring a nucleic acid encoding anADAMTS13 protein in a chemically defined culture medium containing atleast at or about 2 μM zinc. In another embodiment, the method comprisesculturing a cell harboring a nucleic acid encoding an ADAMTS13 proteinin a chemically defined culture medium comprising at least at or about 5μM zinc. In one embodiment, the chemically defined culture mediumcontains at or about between 2 μM and 12 μM zinc. In another embodiment,the chemically defined culture medium contains at or about between 5 μMand 12 μM zinc. In yet other embodiments, the animal protein and/orpolypeptide free culture medium may contain at least at or about 2 μM,or at least at or about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM,11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, or more zinc. Incertain embodiments, the chemically defined medium will be free ofanimal derived proteins and/or polypeptides. In one embodiment, theculture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine.

In one embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a mammalian cell harboring anucleic acid encoding an ADAMTS13 protein in a culture medium comprisingat least at or about 2 μM zinc. In another embodiment, the methodcomprises culturing a mammalian cell harboring a nucleic acid encodingan ADAMTS13 protein in a culture medium comprising at least at or about5 μM zinc. In one embodiment, the method comprises culturing a mammaliancell harboring a nucleic acid encoding an ADAMTS13 protein in a culturemedium comprising at or about between 2 μM and 12 μM zinc. In anotherembodiment, the method comprises culturing a mammalian cell harboring anucleic acid encoding an ADAMTS13 protein in a culture medium comprisingat or about between 5 μM and 12 μM zinc. In yet other embodiments, themethod comprises culturing a mammalian cell harboring a nucleic acidencoding an ADAMTS13 protein in a culture medium comprising at least ator about 2 μM, or at least about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, ormore zinc. In particular embodiments, the mammalian cell is a hamster,human, or murine cell. In a specific embodiment, the cell is a CHO cellline, an HEK 293 cell line, or a BHK cell line. In another embodiment,the mammalian cell is cultured in an animal protein and/or polypeptidefree culture medium. In yet another embodiment, the mammalian cell iscultured in a synthetic culture medium, which may or may not be free ofanimal proteins and polypeptides. In one embodiment, the culture mediumfurther comprises a polyamine at or about between 0.5 mg/L and 30 mg/L.In another embodiment, the culture medium further comprises a polyamineat or about between 2 mg/L and 8 mg/L. In a specific embodiment, thepolyamine is putrescine.

In one embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 2 μM zinc, under continuous or fed-batch cultureconditions. In another embodiment, the method comprises culturing amammalian cell harboring a nucleic acid encoding an ADAMTS13 protein ina culture medium comprising at least at or about 5 μM zinc, undercontinuous or fed-batch culture conditions. In one embodiment, themethod comprises culturing a mammalian cell harboring a nucleic acidencoding an ADAMTS13 protein in a culture medium comprising at or aboutbetween 2 μM and 12 μM zinc, under continuous or fed-batch cultureconditions. In another embodiment, the method comprises culturing amammalian cell harboring a nucleic acid encoding an ADAMTS13 protein ina culture medium comprising at or about between 5 μM and 12 μM zinc,under continuous or fed-batch culture conditions. In yet otherembodiments, the method comprises culturing a mammalian cell harboring anucleic acid encoding an ADAMTS13 protein in a culture medium comprisingat least at or about 2 μM, or at least at or about 3 μM, 4 μM, 5 μM, 6μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM,25 μM, 30 μM, or more zinc, under continuous or fed-batch cultureconditions. In a specific embodiment, the continuous culture conditionis a chemostatic culture condition. In another specific embodiment, thecontinuous culture condition is a perfusion culture condition. In oneembodiment, the cell harboring the nucleic acid encoding an ADAMTS13protein is a mammalian cell. In particular embodiments, the mammaliancell is a hamster, human, or murine cell. In a specific embodiment, thecell is a CHO cell line, an HEK 293 cell line, or a BHK cell line. Inanother embodiment, the mammalian cell is cultured in an animal proteinand/or polypeptide free culture medium. In yet another embodiment, themammalian cell is cultured in a synthetic culture medium, which may ormay not be free of animal proteins and polypeptides. In one embodiment,the culture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 2 μM zinc, wherein the culture is maintained at orabout between 35° C. and 37° C. In another embodiment, the methodcomprises culturing a mammalian cell harboring a nucleic acid encodingan ADAMTS13 protein in a culture medium comprising at least at or about5 μM zinc, wherein the culture is maintained at or about between 35° C.and 37° C. In one embodiment, the method comprises culturing a mammaliancell harboring a nucleic acid encoding an ADAMTS13 protein in a culturemedium comprising at or about between 2 μM and 12 μM zinc, wherein theculture is maintained at or about between 35° C. and 37° C. In anotherembodiment, the method comprises culturing a mammalian cell harboring anucleic acid encoding an ADAMTS13 protein in a culture medium comprisingat or about between 5 μM and 12 μM zinc, wherein the culture ismaintained at or about between 35° C. and 37° C. In yet otherembodiments, the method comprises culturing a mammalian cell harboring anucleic acid encoding an ADAMTS13 protein in a culture medium comprisingat least at or about 2 μM, or at least at or about 3 μM, 4 μM, 5 μM, 6μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM,25 μM, 30 μM, or more zinc, wherein the culture is maintained at orabout between 35° C. and 37° C. In a specific embodiment, the culture ismaintained at or about 36° C. In one embodiment, the temperature of theculture is maintained for at least 7 days. In one embodiment, the methodis performed is under continuous or fed-batch culture conditions. In aspecific embodiment, the continuous culture condition is a chemostaticculture condition. In another specific embodiment, the continuousculture condition is a perfusion culture condition. In one embodiment,the cell harboring the nucleic acid encoding an ADAMTS13 protein is amammalian cell. In particular embodiments, the mammalian cell is ahamster, human, or murine cell. In a specific embodiment, the cell is aCHO cell line, an HEK 293 cell line, or a BHK cell line. In anotherembodiment, the mammalian cell is cultured in an animal protein and/orpolypeptide free culture medium. In yet another embodiment, themammalian cell is cultured in a synthetic culture medium, which may ormay not be free of animal proteins and polypeptides. In one embodiment,the culture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 2 μM zinc, wherein the pH of the culture is maintainedat or about between 6.9 and 7.3. In another embodiment, the methodcomprises culturing a mammalian cell harboring a nucleic acid encodingan ADAMTS13 protein in a culture medium comprising at least at or about5 μM zinc, wherein the pH of the culture is maintained at or aboutbetween 6.9 and 7.3. In one embodiment, the method comprises culturing amammalian cell harboring a nucleic acid encoding an ADAMTS13 protein ina culture medium comprising at or about between 2 μM and 12 μM zinc,wherein the pH of the culture is maintained at or about between 6.9 and7.3. In another embodiment, the method comprises culturing a mammaliancell harboring a nucleic acid encoding an ADAMTS13 protein in a culturemedium comprising at or about between 5 μM and 12 μM zinc, wherein thepH of the culture is maintained at or about between 6.9 and 7.3. In yetother embodiments, the method comprises culturing a mammalian cellharboring a nucleic acid encoding an ADAMTS13 protein in a culturemedium comprising at least at or about 2 μM, or at least at or about 3μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14μM, 15 μM, 20 μM, 25 μM, 30 μM, or more zinc, wherein the pH of theculture is maintained at or about between 6.9 and 7.3. In oneembodiment, the culture is maintained at or about between 35° C. and 37°C. In a specific embodiment, the culture is maintained at or about 36°C. In one embodiment, the temperature and/or pH of the culture ismaintained for at least 7 days. In one embodiment, the method isperformed is under continuous or fed-batch culture conditions. In aspecific embodiment, the continuous culture condition is a chemostaticculture condition. In another specific embodiment, the continuousculture condition is a perfusion culture condition. In one embodiment,the cell harboring the nucleic acid encoding an ADAMTS13 protein is amammalian cell. In particular embodiments, the mammalian cell is ahamster, human, or murine cell. In a specific embodiment, the cell is aCHO cell line, an HEK 293 cell line, or a BHK cell line. In anotherembodiment, the mammalian cell is cultured in an animal protein and/orpolypeptide free culture medium. In yet another embodiment, themammalian cell is cultured in a synthetic culture medium, which may ormay not be free of animal proteins and polypeptides. In one embodiment,the culture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine.

In one embodiment, culture medium used for the expression of an ADAMTSprotein may be supplemented with zinc at a final concentration of atleast about 2 μM to at least about 12 μM. In certain embodiments, theculture medium may be supplemented with zinc at a final concentration ofat least about 2 μM, or at least about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM,or higher levels of zinc. Generally, any zinc salt may be used tosupplement the mediums of the invention, non-limiting examples ofacceptable salts include, ZnSO₄.7H₂O, ZnSO₃.2H₂O, (C₆H₅O₇)₂Zn₃.2H₂O,ZnBr₂, ZnBr₂.2H₂O, ZnCl₂, Zn (NO₃)₂.6H₂O, Zn (H₂PO₄)₂.H₂O,(C₂H₃O₂)₂Zn.2H₂O, and the like. In certain embodiments, apharmaceutically acceptable salt of zinc is used to supplement theculture mediums of the invention.

2. Calcium Supplementation

Advantageously, it was found that increased ADAMTS13 enzymatic activityand specific activity could be recovered from a cell culture grown in amedium supplemented with calcium. Traditional cell culture mediums,e.g., DMEM/F12, typically contained about 1 mM calcium. Historically,these high calcium levels were introduced into the medium to assist withadherent cell cultures. Nowadays, however, commercially availablemediums designed for suspension cultures contain significantly lowercalcium levels, e.g., about 0.1 mM calcium. Such lower calcium levelsare relied upon to prevent aggregation of cells cultured via suspensionmethods. It is known that lower calcium levels are sufficient forpropagating cells in suspension and expressing recombinant proteins,however, the inventors have found that these low calcium levels areinsufficient for the expression of the ADAMTS proteins (e.g., ADAMTS13).

Accordingly, in one aspect, the present invention provides methods forexpressing an ADAMTS protein (e.g., ADAMTS13) having an increasedspecific activity by culturing a cell harboring a nucleic acid encodingan ADAMTS protein (e.g., ADAMTS13) in a culture medium supplemented withcalcium, for example, containing at least 0.5 mM calcium. Similarly, thepresent invention also provides methods for preparing an ADAMTS proteincomposition (e.g., an ADAMTS13 composition) having increased totalactivity or specific activity by culturing a cell harboring a nucleicacid encoding an ADAMTS protein (e.g., ADAMTS13) in a culture mediumsupplemented with calcium, for example, containing at least 0.5 mMcalcium.

In one embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 0.5 mM calcium. In another embodiment, the methodcomprises culturing a cell harboring a nucleic acid encoding an ADAMTS13protein in a culture medium comprising at least at or about 1.5 mMcalcium. In one embodiment, the culture medium contains at or aboutbetween 0.5 mM and 1.5 mM calcium. In yet other embodiments, the culturemedium may contain at least at or about 0.5 mM, or at least at or about0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM,1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, or more calcium.

In one embodiment, a method is provided for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein,comprising culturing a cell harboring a nucleic acid encoding anADAMTS13 protein in an animal protein and/or polypeptide free culturemedium containing at least at or about 0.5 mM calcium. In anotherembodiment, the method comprises culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in an animal protein and/orpolypeptide free culture medium comprising at least at or about 1.5 mMcalcium. In one embodiment, the animal protein and/or polypeptide freeculture medium contains at or about between 0.5 mM and 1.5 mM calcium.In yet other embodiments, the animal protein and/or polypeptide freeculture medium may contain at least at or about 0.5 mM, or at least ator about 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM,1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM,2.75 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, or more calcium. In oneembodiment, the culture medium further comprises a polyamine at or aboutbetween 0.5 mg/L and 30 mg/L. In another embodiment, the culture mediumfurther comprises a polyamine at or about between 2 mg/L and 8 mg/L. Ina specific embodiment, the polyamine is putrescine.

In another embodiment, a method is provided for expressing a disintegrinand metalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein,comprising culturing a cell harboring a nucleic acid encoding anADAMTS13 protein in a chemically defined culture medium containing atleast at or about 0.5 mM calcium. In another embodiment, the methodcomprises culturing a cell harboring a nucleic acid encoding an ADAMTS13protein in a chemically defined culture medium comprising at least at orabout 1.5 mM calcium. In one embodiment, the chemically defined culturemedium contains at or about between 0.5 mM and 1.5 mM calcium. In yetother embodiments, the animal protein and/or polypeptide free culturemedium may contain at least at or about 0.5 mM, or at least at or about0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM,1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, or more calcium. In certainembodiments, the chemically defined medium will be free of animalderived proteins and/or polypeptides. In one embodiment, the culturemedium further comprises a polyamine at or about between 0.5 mg/L and 30mg/L. In another embodiment, the culture medium further comprises apolyamine at or about between 2 mg/L and 8 mg/L. In a specificembodiment, the polyamine is putrescine.

In one embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a mammalian cell harboring anucleic acid encoding an ADAMTS13 protein in a culture medium comprisingat least at or about 0.5 mM calcium. In another embodiment, the methodcomprises culturing a mammalian cell harboring a nucleic acid encodingan ADAMTS13 protein in a culture medium comprising at least at or about1.5 mM calcium. In one embodiment, the method comprises culturing amammalian cell harboring a nucleic acid encoding an ADAMTS13 protein ina culture medium comprising at or about between 0.5 mM and 1.5 mMcalcium. In yet other embodiments, the method comprises culturing amammalian cell harboring a nucleic acid encoding an ADAMTS13 protein ina culture medium comprising at least at or about 0.5 mM, or at least ator about 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM,1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM,2.75 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, or more calcium. Inparticular embodiments, the mammalian cell is a hamster, human, ormurine cell. In a specific embodiment, the cell is a CHO cell line, anHEK 293 cell line, or a BHK cell line. In another embodiment, themammalian cell is cultured in an animal protein and/or polypeptide freeculture medium. In yet another embodiment, the mammalian cell iscultured in a synthetic culture medium, which may or may not be free ofanimal proteins and polypeptides. In one embodiment, the culture mediumfurther comprises a polyamine at or about between 0.5 mg/L and 30 mg/L.In another embodiment, the culture medium further comprises a polyamineat or about between 2 mg/L and 8 mg/L. In a specific embodiment, thepolyamine is putrescine.

In one embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 0.5 mM calcium, under continuous or fed-batch cultureconditions. In another embodiment, the method comprises culturing amammalian cell harboring a nucleic acid encoding an ADAMTS13 protein ina culture medium comprising at least at or about 1.5 mM calcium, undercontinuous or fed-batch culture conditions. In one embodiment, themethod comprises culturing a mammalian cell harboring a nucleic acidencoding an ADAMTS13 protein in a culture medium comprising at or aboutbetween 0.5 mM and 1.5 mM calcium, under continuous or fed-batch cultureconditions. In yet other embodiments, the method comprises culturing amammalian cell harboring a nucleic acid encoding an ADAMTS13 protein ina culture medium comprising at least at or about 0.5 mM, or at least ator about 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM,1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM,2.75 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, or more calcium, undercontinuous or fed-batch culture conditions. In a specific embodiment,the continuous culture condition is a chemostatic culture condition. Inanother specific embodiment, the continuous culture condition is aperfusion culture condition. In one embodiment, the cell harboring thenucleic acid encoding an ADAMTS13 protein is a mammalian cell. Inparticular embodiments, the mammalian cell is a hamster, human, ormurine cell. In a specific embodiment, the cell is a CHO cell line, anHEK 293 cell line, or a BHK cell line. In another embodiment, themammalian cell is cultured in an animal protein and/or polypeptide freeculture medium. In yet another embodiment, the mammalian cell iscultured in a synthetic culture medium, which may or may not be free ofanimal proteins and polypeptides. In one embodiment, the culture mediumfurther comprises a polyamine at or about between 0.5 mg/L and 30 mg/L.In another embodiment, the culture medium further comprises a polyamineat or about between 2 mg/L and 8 mg/L. In a specific embodiment, thepolyamine is putrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 0.5 mM calcium, wherein the culture is maintained ator about between 35° C. and 37° C. In another embodiment, the methodcomprises culturing a mammalian cell harboring a nucleic acid encodingan ADAMTS13 protein in a culture medium comprising at least at or about1.5 mM calcium, wherein the culture is maintained at or about between35° C. and 37° C. In one embodiment, the method comprises culturing amammalian cell harboring a nucleic acid encoding an ADAMTS13 protein ina culture medium comprising at or about between 0.5 mM and 1.5 mMcalcium, wherein the culture is maintained at or about between 35° C.and 37° C. In yet other embodiments, the method comprises culturing amammalian cell harboring a nucleic acid encoding an ADAMTS13 protein ina culture medium comprising at least at or about 0.5 mM, or at least ator about 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM,1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM,2.75 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, or more calcium,wherein the culture is maintained at or about between 35° C. and 37° C.In a specific embodiment, the culture is maintained at or about 36° C.In one embodiment, the temperature of the culture is maintained for atleast 7 days. In one embodiment, the method is performed is undercontinuous or fed-batch culture conditions. In a specific embodiment,the continuous culture condition is a chemostatic culture condition. Inanother specific embodiment, the continuous culture condition is aperfusion culture condition. In one embodiment, the cell harboring thenucleic acid encoding an ADAMTS13 protein is a mammalian cell. Inparticular embodiments, the mammalian cell is a hamster, human, ormurine cell. In a specific embodiment, the cell is a CHO cell line, anHEK 293 cell line, or a BHK cell line. In another embodiment, themammalian cell is cultured in an animal protein and/or polypeptide freeculture medium. In yet another embodiment, the mammalian cell iscultured in a synthetic culture medium, which may or may not be free ofanimal proteins and polypeptides. In one embodiment, the culture mediumfurther comprises a polyamine at or about between 0.5 mg/L and 30 mg/L.In another embodiment, the culture medium further comprises a polyamineat or about between 2 mg/L and 8 mg/L. In a specific embodiment, thepolyamine is putrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 0.5 mM calcium, wherein the pH of the culture ismaintained at or about between 6.9 and 7.3. In another embodiment, themethod comprises culturing a mammalian cell harboring a nucleic acidencoding an ADAMTS13 protein in a culture medium comprising at least ator about 1.5 mM calcium, wherein the pH of the culture is maintained ator about between 6.9 and 7.3. In one embodiment, the method comprisesculturing a mammalian cell harboring a nucleic acid encoding an ADAMTS13protein in a culture medium comprising at or about between 0.5 mM and1.5 mM calcium, wherein the pH of the culture is maintained at or aboutbetween 6.9 and 7.3. In yet other embodiments, the method comprisesculturing a mammalian cell harboring a nucleic acid encoding an ADAMTS13protein in a culture medium comprising at least at or about 0.5 mM, orat least at or about 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, or morecalcium, wherein the pH of the culture is maintained at or about between6.9 and 7.3. In one embodiment, the culture is maintained at or aboutbetween 35° C. and 37° C. In a specific embodiment, the culture ismaintained at or about 36° C. In one embodiment, the temperature and/orpH of the culture is maintained for at least 7 days. In one embodiment,the method is performed is under continuous or fed-batch cultureconditions. In a specific embodiment, the continuous culture conditionis a chemostatic culture condition. In another specific embodiment, thecontinuous culture condition is a perfusion culture condition. In oneembodiment, the cell harboring the nucleic acid encoding an ADAMTS13protein is a mammalian cell. In particular embodiments, the mammaliancell is a hamster, human, or murine cell. In a specific embodiment, thecell is a CHO cell line, an HEK 293 cell line, or a BHK cell line. Inanother embodiment, the mammalian cell is cultured in an animal proteinand/or polypeptide free culture medium. In yet another embodiment, themammalian cell is cultured in a synthetic culture medium, which may ormay not be free of animal proteins and polypeptides. In one embodiment,the culture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 0.5 mM calcium and at least at or about 2 μM zinc. Ina related embodiment, the culture medium comprises at least at or about0.5 mM calcium and at least at or about 5 μM zinc. In another relatedembodiment, the culture medium comprises at least at or about 0.5 mMcalcium and at least at or about between 2 μM zinc and 12 μM zinc. Inyet another related embodiment, the culture medium comprises at least ator about 0.5 mM calcium and at least at or about between 5 μM zinc and12 μM zinc. In certain embodiments, the culture medium comprises atleast at or about 0.5 mM calcium and at least at or about 2 μM, or atleast at or about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, or more zinc. Inone embodiment, the culture is maintained at or about between 35° C. and37° C. In a specific embodiment, the culture is maintained at or about36° C. In one embodiment, the temperature and/or pH of the culture ismaintained for at least 7 days. In one embodiment, the method isperformed is under continuous or fed-batch culture conditions. In aspecific embodiment, the continuous culture condition is a chemostaticculture condition. In another specific embodiment, the continuousculture condition is a perfusion culture condition. In one embodiment,the cell harboring the nucleic acid encoding an ADAMTS13 protein is amammalian cell. In particular embodiments, the mammalian cell is ahamster, human, or murine cell. In a specific embodiment, the cell is aCHO cell line, an HEK 293 cell line, or a BHK cell line. In anotherembodiment, the mammalian cell is cultured in an animal protein and/orpolypeptide free culture medium. In yet another embodiment, themammalian cell is cultured in a synthetic culture medium, which may ormay not be free of animal proteins and polypeptides. In one embodiment,the culture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 1.5 mM calcium and at least at or about 2 μM zinc. Ina related embodiment, the culture medium comprises at least at or about1.5 mM calcium and at least at or about 5 μM zinc. In another relatedembodiment, the culture medium comprises at least at or about 1.5 mMcalcium and at least at or about between 2 μM zinc and 12 μM zinc. Inyet another related embodiment, the culture medium comprises at least ator about 1.5 mM calcium and at least at or about between 5 μM zinc and12 μM zinc. In certain embodiments, the culture medium comprises atleast at or about 1.5 mM calcium and at least at or about 2 μM, or atleast at or about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, or more zinc. Inone embodiment, the culture is maintained at or about between 35° C. and37° C. In a specific embodiment, the culture is maintained at or about36° C. In one embodiment, the temperature and/or pH of the culture ismaintained for at least 7 days. In one embodiment, the method isperformed is under continuous or fed-batch culture conditions. In aspecific embodiment, the continuous culture condition is a chemostaticculture condition. In another specific embodiment, the continuousculture condition is a perfusion culture condition. In one embodiment,the cell harboring the nucleic acid encoding an ADAMTS13 protein is amammalian cell. In particular embodiments, the mammalian cell is ahamster, human, or murine cell. In a specific embodiment, the cell is aCHO cell line, an HEK 293 cell line, or a BHK cell line. In anotherembodiment, the mammalian cell is cultured in an animal protein and/orpolypeptide free culture medium. In yet another embodiment, themammalian cell is cultured in a synthetic culture medium, which may ormay not be free of animal proteins and polypeptides. In one embodiment,the culture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about between 0.5 mM and 1.5 mM calcium and at least at orabout 2 μM zinc. In a related embodiment, the culture medium comprisesat least at or about between 0.5 mM and 1.5 mM calcium and at least ator about 5 μM zinc. In another related embodiment, the culture mediumcomprises at least at or about between 0.5 mM and 1.5 mM calcium and atleast at or about between 2 μM zinc and 12 μM zinc. In yet anotherrelated embodiment, the culture medium comprises at least at or aboutbetween 0.5 mM and 1.5 mM calcium and at least at or about between 5 μMzinc and 12 μM zinc. In certain embodiments, the culture mediumcomprises at least at or about between 0.5 mM and 1.5 mM calcium and atleast at or about 2 μM, or at least at or about 3 μM, 4 μM, 5 μM, 6 μM,7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25μM, 30 μM, or more zinc. In one embodiment, the culture is maintained ator about between 35° C. and 37° C. In a specific embodiment, the cultureis maintained at or about 36° C. In one embodiment, the temperatureand/or pH of the culture is maintained for at least 7 days. In oneembodiment, the method is performed is under continuous or fed-batchculture conditions. In a specific embodiment, the continuous culturecondition is a chemostatic culture condition. In another specificembodiment, the continuous culture condition is a perfusion culturecondition. In one embodiment, the cell harboring the nucleic acidencoding an ADAMTS13 protein is a mammalian cell. In particularembodiments, the mammalian cell is a hamster, human, or murine cell. Ina specific embodiment, the cell is a CHO cell line, an HEK 293 cellline, or a BHK cell line. In another embodiment, the mammalian cell iscultured in an animal protein and/or polypeptide free culture medium. Inyet another embodiment, the mammalian cell is cultured in a syntheticculture medium, which may or may not be free of animal proteins andpolypeptides. In one embodiment, the culture medium further comprises apolyamine at or about between 0.5 mg/L and 30 mg/L. In anotherembodiment, the culture medium further comprises a polyamine at or aboutbetween 2 mg/L and 8 mg/L. In a specific embodiment, the polyamine isputrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 0.5 mM, or at least at or about 0.6 mM, 0.7 mM, 0.8mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5 mM,4.0 mM, 4.5 mM, 5.0 mM, or more calcium and at least at or about 2 μMzinc. In a related embodiment, the culture medium comprises at or about0.5 mM, or at least at or about 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM,1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM,2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0mM, or more calcium and at least at or about 5 μM zinc. In anotherrelated embodiment, the culture medium comprises at or about 0.5 mM, orat least at or about 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, or morecalcium and at least at or about between 2 μM zinc and 12 μM zinc. Inyet another related embodiment, the culture medium comprises at or about0.5 mM, or at least at or about 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM,1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM,2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0mM, or more calcium and at least at or about between 5 μM zinc and 12 μMzinc. In certain embodiments, the culture medium comprises at or about0.5 mM, or at least at or about 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM,1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM,2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0mM, or more calcium and at least at or about 2 μM, or at least at orabout 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, or more zinc. In one embodiment,the culture is maintained at or about between 35° C. and 37° C. In aspecific embodiment, the culture is maintained at or about 36° C. In oneembodiment, the temperature and/or pH of the culture is maintained forat least 7 days. In one embodiment, the method is performed is undercontinuous or fed-batch culture conditions. In a specific embodiment,the continuous culture condition is a chemostatic culture condition. Inanother specific embodiment, the continuous culture condition is aperfusion culture condition. In one embodiment, the cell harboring thenucleic acid encoding an ADAMTS13 protein is a mammalian cell. Inparticular embodiments, the mammalian cell is a hamster, human, ormurine cell. In a specific embodiment, the cell is a CHO cell line, anHEK 293 cell line, or a BHK cell line. In another embodiment, themammalian cell is cultured in an animal protein and/or polypeptide freeculture medium. In yet another embodiment, the mammalian cell iscultured in a synthetic culture medium, which may or may not be free ofanimal proteins and polypeptides. In one embodiment, the culture mediumfurther comprises a polyamine at or about between 0.5 mg/L and 30 mg/L.In another embodiment, the culture medium further comprises a polyamineat or about between 2 mg/L and 8 mg/L. In a specific embodiment, thepolyamine is putrescine.

Generally, any calcium salt may be used to supplement the mediums of theinvention. Non-limiting examples of acceptable salts include CaCl₂,CaCl₂, CaFPO₃.2H₂O, CaI₂, CaBr₂, (C₂H₃O₂)₂Ca, (CHO₂)₂Ca, (C₆H₇O₆)₂Ca,(C₆H₅O₇)₂Ca₃.2H₂O, and the like. In certain embodiments, apharmaceutically acceptable salt of calcium is used to supplement theculture mediums of the invention.

3. Nicotinamide (Vitamin B3) Supplementation

Advantageously, it was found that increased ADAMTS13 enzymatic activityand specific activity could be recovered from a cell culture grown in amedium supplemented with nicotinamide (vitamin B3). For example, Example2, demonstrates that ADAMTS13 protein expressed in culture mediumcontaining 7 mg/L nicotinamide (vitamin B3) has a 60% higher specificactivity than ADAMTS13 protein expressed in culture medium containingonly 2 mg/L nicotinamide (Table 13; compare days 4 and 7 with day 11).Surprisingly, this effect is synergistic with zinc supplementation, asexpression of ADAMTS13 in medium containing 7 mg/L nicotinamide (vitaminB3) and 5 μM zinc results in a 200% to 300% increase in the specificactivity of ADAMTS13 protein (compare Table 14 day 11 with Table 13 days4 and 7).

Accordingly, in one aspect, the present invention provides methods forexpressing an ADAMTS protein (e.g., ADAMTS13) having an increasedspecific activity by culturing a cell harboring a nucleic acid encodingan ADAMTS protein (e.g., ADAMTS13) in a culture medium supplemented withnicotinamide (vitamin B3), for example, containing at least 2 mg/Lnicotinamide (vitamin B3). Similarly, the present invention alsoprovides methods for preparing an ADAMTS protein composition (e.g., anADAMTS13 composition) having increased total activity or specificactivity by culturing a cell harboring a nucleic acid encoding an ADAMTSprotein (e.g., ADAMTS13) in a culture medium supplemented withnicotinamide (vitamin B3), for example, containing at least 2 mg/Lnicotinamide (vitamin B3).

In one embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 2 mg/L nicotinamide (vitamin B3). In anotherembodiment, the method comprises culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 7 mg/L nicotinamide (vitamin B3). In one embodiment,the culture medium contains at or about between 2 mg/L and 10 mg/Lnicotinamide (vitamin B3). In yet other embodiments, the culture mediummay contain at least at or about 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L,7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or higherconcentrations of nicotinamide (vitamin B3). Suitable nicotinamide(vitamin B3) concentration ranges are generally determined by cellculture toxicities that may occur in the presence of high concentrationsof nicotinamide (vitamin B3), e.g., at concentrations greater than 15mg/L, 20 mg/L, 30 mg/L, 40 mg/L, and the like. As will be understood bythe skilled artisan, the extent to which nicotinamide (vitamin B3)concentrations are inhibitory to a particular culture system will behighly dependent upon, among other factors, the type of cell used toexpress an ADAMTS protein, the components of the culture mediumutilized, and the operative mode employed for the culture (e.g., batchvs. continuous; suspension vs. adherent; chemostat vs. perfusion; etc.).In certain instances, higher nicotinamide (vitamin B3) concentrationsmay be required where components of the culture medium may sequesternicotinamide (vitamin B3) from the solution. Accordingly, suitablenicotinamide (vitamin B3) concentration ranges are generally determinedby the identity of the cultured cells, medium and operative modeemployed. One of skill will readily be able to determine appropriateupper limits for the use of nicotinamide (vitamin B3) supplementationbased on the individual culture system employed.

In one embodiment, a method is provided for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein,comprising culturing a cell harboring a nucleic acid encoding anADAMTS13 protein in an animal protein and/or polypeptide free culturemedium containing at least at or about 2 mg/L nicotinamide (vitamin B3).In another embodiment, the method comprises culturing a cell harboring anucleic acid encoding an ADAMTS13 protein in an animal protein and/orpolypeptide free culture medium comprising at least at or about 7 mg/Lnicotinamide (vitamin B3). In one embodiment, the animal protein and/orpolypeptide free culture medium contains at or about between 2 mg/L and10 mg/L nicotinamide (vitamin B3). In yet other embodiments, the animalprotein and/or polypeptide free culture medium may contain at least ator about 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L, 7 mg/L, 8 mg/L, 9 mg/L,10 mg/L, 15 mg/L, 20 mg/L, or higher concentrations of nicotinamide(vitamin B3). In one embodiment, the culture medium further comprises apolyamine at or about between 0.5 mg/L and 30 mg/L. In anotherembodiment, the culture medium further comprises a polyamine at or aboutbetween 2 mg/L and 8 mg/L. In a specific embodiment, the polyamine isputrescine.

In another embodiment, a method is provided for expressing a disintegrinand metalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein,comprising culturing a cell harboring a nucleic acid encoding anADAMTS13 protein in a chemically defined culture medium containing atleast at or about 2 mg/L nicotinamide (vitamin B3). In anotherembodiment, the method comprises culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a chemically defined culture mediumcomprising at least at or about 7 mg/L nicotinamide (vitamin B3). In oneembodiment, the chemically defined culture medium contains at or aboutbetween 2 mg/L and 10 mg/L nicotinamide (vitamin B3). In yet otherembodiments, the animal protein and/or polypeptide free culture mediummay contain at least at or about 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L,7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or higherconcentrations of nicotinamide (vitamin B3). In certain embodiments, thechemically defined medium will be free of animal derived proteins and/orpolypeptides. In one embodiment, the culture medium further comprises apolyamine at or about between 0.5 mg/L and 30 mg/L. In anotherembodiment, the culture medium further comprises a polyamine at or aboutbetween 2 mg/L and 8 mg/L. In a specific embodiment, the polyamine isputrescine.

In one embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a mammalian cell harboring anucleic acid encoding an ADAMTS13 protein in a culture medium comprisingat least at or about 2 mg/L nicotinamide (vitamin B3). In anotherembodiment, the method comprises culturing a mammalian cell harboring anucleic acid encoding an ADAMTS13 protein in a culture medium comprisingat least at or about 7 mg/L nicotinamide (vitamin B3). In oneembodiment, the method comprises culturing a mammalian cell harboring anucleic acid encoding an ADAMTS13 protein in a culture medium comprisingat or about between 2 mg/L and 10 mg/L nicotinamide (vitamin B3). In yetother embodiments, the method comprises culturing a mammalian cellharboring a nucleic acid encoding an ADAMTS13 protein in a culturemedium comprising at least at or about 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or higherconcentrations of nicotinamide (vitamin B3). In particular embodiments,the mammalian cell is a hamster, human, or murine cell. In a specificembodiment, the cell is a CHO cell line, an HEK 293 cell line, or a BHKcell line. In another embodiment, the mammalian cell is cultured in ananimal protein and/or polypeptide free culture medium. In yet anotherembodiment, the mammalian cell is cultured in a synthetic culturemedium, which may or may not be free of animal proteins andpolypeptides. In one embodiment, the culture medium further comprises apolyamine at or about between 0.5 mg/L and 30 mg/L. In anotherembodiment, the culture medium further comprises a polyamine at or aboutbetween 2 mg/L and 8 mg/L. In a specific embodiment, the polyamine isputrescine.

In one embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 2 mg/L nicotinamide (vitamin B3), under continuous orfed-batch culture conditions. In another embodiment, the methodcomprises culturing a mammalian cell harboring a nucleic acid encodingan ADAMTS13 protein in a culture medium comprising at least at or about7 mg/L nicotinamide (vitamin B3), under continuous or fed-batch cultureconditions. In one embodiment, the method comprises culturing amammalian cell harboring a nucleic acid encoding an ADAMTS13 protein ina culture medium comprising at or about between 2 mg/L and 10 mg/Lnicotinamide (vitamin B3), under continuous or fed-batch cultureconditions. In yet other embodiments, the method comprises culturing amammalian cell harboring a nucleic acid encoding an ADAMTS13 protein ina culture medium comprising at least at or about 2 mg/L, 3 mg/L, 4 mg/L,5 mg/L, 6 mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, orhigher concentrations of nicotinamide (vitamin B3), under continuous orfed-batch culture conditions. In a specific embodiment, the continuousculture condition is a chemostatic culture condition. In anotherspecific embodiment, the continuous culture condition is a perfusionculture condition. In one embodiment, the cell harboring the nucleicacid encoding an ADAMTS13 protein is a mammalian cell. In particularembodiments, the mammalian cell is a hamster, human, or murine cell. Ina specific embodiment, the cell is a CHO cell line, an HEK 293 cellline, or a BHK cell line. In another embodiment, the mammalian cell iscultured in an animal protein and/or polypeptide free culture medium. Inyet another embodiment, the mammalian cell is cultured in a syntheticculture medium, which may or may not be free of animal proteins andpolypeptides. In one embodiment, the culture medium further comprises apolyamine at or about between 0.5 mg/L and 30 mg/L. In anotherembodiment, the culture medium further comprises a polyamine at or aboutbetween 2 mg/L and 8 mg/L. In a specific embodiment, the polyamine isputrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 2 mg/L nicotinamide (vitamin B3), wherein the cultureis maintained at or about between 35° C. and 37° C. In anotherembodiment, the method comprises culturing a mammalian cell harboring anucleic acid encoding an ADAMTS13 protein in a culture medium comprisingat least at or about 7 mg/L nicotinamide (vitamin B3), wherein theculture is maintained at or about between 35° C. and 37° C. In oneembodiment, the method comprises culturing a mammalian cell harboring anucleic acid encoding an ADAMTS13 protein in a culture medium comprisingat or about between 2 mg/L and 10 mg/L nicotinamide, wherein the cultureis maintained at or about between 35° C. and 37° C. In yet otherembodiments, the method comprises culturing a mammalian cell harboring anucleic acid encoding an ADAMTS13 protein in a culture medium comprisingat least at or about 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L, 7 mg/L, 8mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or higher concentrations ofnicotinamide (vitamin B3), wherein the culture is maintained at or aboutbetween 35° C. and 37° C. In a specific embodiment, the culture ismaintained at or about 36° C. In one embodiment, the temperature of theculture is maintained for at least 7 days. In one embodiment, the methodis performed is under continuous or fed-batch culture conditions. In aspecific embodiment, the continuous culture condition is a chemostaticculture condition. In another specific embodiment, the continuousculture condition is a perfusion culture condition. In one embodiment,the cell harboring the nucleic acid encoding an ADAMTS13 protein is amammalian cell. In particular embodiments, the mammalian cell is ahamster, human, or murine cell. In a specific embodiment, the cell is aCHO cell line, an HEK 293 cell line, or a BHK cell line. In anotherembodiment, the mammalian cell is cultured in an animal protein and/orpolypeptide free culture medium. In yet another embodiment, themammalian cell is cultured in a synthetic culture medium, which may ormay not be free of animal proteins and polypeptides. In one embodiment,the culture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 2 mg/L nicotinamide (vitamin B3), wherein the pH ofthe culture is maintained at or about between 6.9 and 7.3. In anotherembodiment, the method comprises culturing a mammalian cell harboring anucleic acid encoding an ADAMTS13 protein in a culture medium comprisingat least at or about 7 mg/L nicotinamide (vitamin B3), wherein the pH ofthe culture is maintained at or about between 6.9 and 7.3. In oneembodiment, the method comprises culturing a mammalian cell harboring anucleic acid encoding an ADAMTS13 protein in a culture medium comprisingat or about between 2 mg/L and 10 mg/L nicotinamide (vitamin B3),wherein the pH of the culture is maintained at or about between 6.9 and7.3. In yet other embodiments, the method comprises culturing amammalian cell harboring a nucleic acid encoding an ADAMTS13 protein ina culture medium comprising at least at or about 2 mg/L, 3 mg/L, 4 mg/L,5 mg/L, 6 mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, orhigher concentrations of nicotinamide (vitamin B3), wherein the pH ofthe culture is maintained at or about between 6.9 and 7.3. In oneembodiment, the culture is maintained at or about between 35° C. and 37°C. In a specific embodiment, the culture is maintained at or about 36°C. In one embodiment, the temperature and/or pH of the culture ismaintained for at least 7 days. In one embodiment, the method isperformed is under continuous or fed-batch culture conditions. In aspecific embodiment, the continuous culture condition is a chemostaticculture condition. In another specific embodiment, the continuousculture condition is a perfusion culture condition. In one embodiment,the cell harboring the nucleic acid encoding an ADAMTS13 protein is amammalian cell. In particular embodiments, the mammalian cell is ahamster, human, or murine cell. In a specific embodiment, the cell is aCHO cell line, an HEK 293 cell line, or a BHK cell line. In anotherembodiment, the mammalian cell is cultured in an animal protein and/orpolypeptide free culture medium. In yet another embodiment, themammalian cell is cultured in a synthetic culture medium, which may ormay not be free of animal proteins and polypeptides. In one embodiment,the culture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 2 mg/L nicotinamide (vitamin B3) and at least at orabout 2 μM zinc. In a related embodiment, the culture medium comprisesat least at or about 2 mg/L nicotinamide (vitamin B3) and at least at orabout 5 μM zinc. In another related embodiment, the culture mediumcomprises at least at or about 2 mg/L nicotinamide (vitamin B3) and atleast at or about between 2 μM zinc and 12 μM zinc. In yet anotherrelated embodiment, the culture medium comprises at least at or about 2mg/L nicotinamide (vitamin B3) and at least at or about between 5 μMzinc and 12 μM zinc. In certain embodiments, the culture mediumcomprises at least at or about 2 mg/L nicotinamide (vitamin B3) and atleast at or about 2 μM, or at least at or about 3 μM, 4 μM, 5 μM, 6 μM,7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25μM, 30 μM, or more zinc. In one embodiment, the culture is maintained ator about between 35° C. and 37° C. In a specific embodiment, the cultureis maintained at or about 36° C. In one embodiment, the temperatureand/or pH of the culture is maintained for at least 7 days. In oneembodiment, the method is performed is under continuous or fed-batchculture conditions. In a specific embodiment, the continuous culturecondition is a chemostatic culture condition. In another specificembodiment, the continuous culture condition is a perfusion culturecondition. In one embodiment, the cell harboring the nucleic acidencoding an ADAMTS13 protein is a mammalian cell. In particularembodiments, the mammalian cell is a hamster, human, or murine cell. Ina specific embodiment, the cell is a CHO cell line, an HEK 293 cellline, or a BHK cell line. In another embodiment, the mammalian cell iscultured in an animal protein and/or polypeptide free culture medium. Inyet another embodiment, the mammalian cell is cultured in a syntheticculture medium, which may or may not be free of animal proteins andpolypeptides. In one embodiment, the culture medium further comprises apolyamine at or about between 0.5 mg/L and 30 mg/L. In anotherembodiment, the culture medium further comprises a polyamine at or aboutbetween 2 mg/L and 8 mg/L. In a specific embodiment, the polyamine isputrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 7 mg/L nicotinamide (vitamin B3) and at least at orabout 2 μM zinc. In a related embodiment, the culture medium comprisesat least at or about 7 mg/L nicotinamide (vitamin B3) and at least at orabout 5 μM zinc. In another related embodiment, the culture mediumcomprises at least at or about 7 mg/L nicotinamide (vitamin B3) and atleast at or about between 2 μM zinc and 12 μM zinc. In yet anotherrelated embodiment, the culture medium comprises at least at or about 7mg/L nicotinamide (vitamin B3) and at least at or about between 5 μMzinc and 12 μM zinc. In certain embodiments, the culture mediumcomprises at least at or about 7 mg/L nicotinamide (vitamin B3) and atleast at or about 2 μM, or at least at or about 3 μM, 4 μM, 5 μM, 6 μM,7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25μM, 30 μM, or more zinc. In one embodiment, the culture is maintained ator about between 35° C. and 37° C. In a specific embodiment, the cultureis maintained at or about 36° C. In one embodiment, the temperatureand/or pH of the culture is maintained for at least 7 days. In oneembodiment, the method is performed is under continuous or fed-batchculture conditions. In a specific embodiment, the continuous culturecondition is a chemostatic culture condition. In another specificembodiment, the continuous culture condition is a perfusion culturecondition. In one embodiment, the cell harboring the nucleic acidencoding an ADAMTS13 protein is a mammalian cell. In particularembodiments, the mammalian cell is a hamster, human, or murine cell. Ina specific embodiment, the cell is a CHO cell line, an HEK 293 cellline, or a BHK cell line. In another embodiment, the mammalian cell iscultured in an animal protein and/or polypeptide free culture medium. Inyet another embodiment, the mammalian cell is cultured in a syntheticculture medium, which may or may not be free of animal proteins andpolypeptides. In one embodiment, the culture medium further comprises apolyamine at or about between 0.5 mg/L and 30 mg/L. In anotherembodiment, the culture medium further comprises a polyamine at or aboutbetween 2 mg/L and 8 mg/L. In a specific embodiment, the polyamine isputrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about between 2 mg/L and 10 mg/L nicotinamide (vitamin B3)and at least at or about 2 μM zinc. In a related embodiment, the culturemedium comprises at least at or about between 2 mg/L and 10 mg/Lnicotinamide (vitamin B3) and at least at or about 5 μM zinc. In anotherrelated embodiment, the culture medium comprises at least at or aboutbetween 2 mg/L and 10 mg/L nicotinamide (vitamin B3) and at least at orabout between 2 μM zinc and 12 μM zinc. In yet another relatedembodiment, the culture medium comprises at least at or about between 2mg/L and 10 mg/L nicotinamide (vitamin B3) and at least at or aboutbetween 5 μM zinc and 12 μM zinc. In certain embodiments, the culturemedium comprises at least at or about between 2 mg/L and 10 mg/Lnicotinamide (vitamin B3) and at least at or about 2 μM, or at least ator about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM,13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, or more zinc. In oneembodiment, the culture is maintained at or about between 35° C. and 37°C. In a specific embodiment, the culture is maintained at or about 36°C. In one embodiment, the temperature and/or pH of the culture ismaintained for at least 7 days. In one embodiment, the method isperformed is under continuous or fed-batch culture conditions. In aspecific embodiment, the continuous culture condition is a chemostaticculture condition. In another specific embodiment, the continuousculture condition is a perfusion culture condition. In one embodiment,the cell harboring the nucleic acid encoding an ADAMTS13 protein is amammalian cell. In particular embodiments, the mammalian cell is ahamster, human, or murine cell. In a specific embodiment, the cell is aCHO cell line, an HEK 293 cell line, or a BHK cell line. In anotherembodiment, the mammalian cell is cultured in an animal protein and/orpolypeptide free culture medium. In yet another embodiment, themammalian cell is cultured in a synthetic culture medium, which may ormay not be free of animal proteins and polypeptides. In one embodiment,the culture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L, 7 mg/L, 8mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or higher concentrations ofnicotinamide (vitamin B3) and at least at or about 2 μM zinc. In arelated embodiment, the culture medium comprises at or about2 mg/L, 3mg/L, 4 mg/L, 5 mg/L, 6 mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L,20 mg/L, or higher concentrations of nicotinamide (vitamin B3) and atleast at or about 5 μM zinc. In another related embodiment, the culturemedium comprises at or about 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L, 7mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or higherconcentrations of nicotinamide (vitamin B3) and at least at or aboutbetween 2 μM zinc and 12 μM zinc. In yet another related embodiment, theculture medium comprises at or about 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or higherconcentrations of nicotinamide (vitamin B3) and at least at or aboutbetween 5 μM zinc and 12 μM zinc. In certain embodiments, the culturemedium comprises at or about 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L, 7mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or higherconcentrations of nicotinamide (vitamin B3) and at least at or about 2μM, or at least at or about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, or morezinc. In one embodiment, the culture is maintained at or about between35° C. and 37° C. In a specific embodiment, the culture is maintained ator about 36° C. In one embodiment, the temperature and/or pH of theculture is maintained for at least 7 days. In one embodiment, the methodis performed is under continuous or fed-batch culture conditions. In aspecific embodiment, the continuous culture condition is a chemostaticculture condition. In another specific embodiment, the continuousculture condition is a perfusion culture condition. In one embodiment,the cell harboring the nucleic acid encoding an ADAMTS13 protein is amammalian cell. In particular embodiments, the mammalian cell is ahamster, human, or murine cell. In a specific embodiment, the cell is aCHO cell line, an HEK 293 cell line, or a BHK cell line. In anotherembodiment, the mammalian cell is cultured in an animal protein and/orpolypeptide free culture medium. In yet another embodiment, themammalian cell is cultured in a synthetic culture medium, which may ormay not be free of animal proteins and polypeptides. In one embodiment,the culture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 2 mg/L nicotinamide (vitamin B3) and at least at orabout 0.5 mM calcium. In a related embodiment, the culture mediumcomprises at least at or about 2 mg/L nicotinamide (vitamin B3) and atleast at or about 1.5 mM calcium. In another related embodiment, theculture medium comprises at least at or about 2 mg/L nicotinamide(vitamin B3) and at least at or about between 0.5 mM and 1.5 mM calcium.In certain embodiments, the culture medium comprises at least at orabout 2 mg/L nicotinamide (vitamin B3) and at least at or about 0.5 mM,or at least at or about 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM,1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM,2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, ormore calcium. In one embodiment, the culture is maintained at or aboutbetween 35° C. and 37° C. In a specific embodiment, the culture ismaintained at or about 36° C. In one embodiment, the temperature and/orpH of the culture is maintained for at least 7 days. In one embodiment,the method is performed is under continuous or fed-batch cultureconditions. In a specific embodiment, the continuous culture conditionis a chemostatic culture condition. In another specific embodiment, thecontinuous culture condition is a perfusion culture condition. In oneembodiment, the cell harboring the nucleic acid encoding an ADAMTS13protein is a mammalian cell. In particular embodiments, the mammaliancell is a hamster, human, or murine cell. In a specific embodiment, thecell is a CHO cell line, an HEK 293 cell line, or a BHK cell line. Inanother embodiment, the mammalian cell is cultured in an animal proteinand/or polypeptide free culture medium. In yet another embodiment, themammalian cell is cultured in a synthetic culture medium, which may ormay not be free of animal proteins and polypeptides. In one embodiment,the culture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 7 mg/L nicotinamide (vitamin B3) and at least at orabout 0.5 mM calcium. In a related embodiment, the culture mediumcomprises at least at or about 7 mg/L nicotinamide (vitamin B3) and atleast at or about 1.5 mM calcium. In another related embodiment, theculture medium comprises at least at or about 7 mg/L nicotinamide(vitamin B3) and at least at or about between 0.5 mM and 1.5 mM calcium.In certain embodiments, the culture medium comprises at least at orabout 7 mg/L nicotinamide (vitamin B3) and at least at or about 0.5 mM,or at least at or about 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM,1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM,2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, ormore calcium. In one embodiment, the culture is maintained at or aboutbetween 35° C. and 37° C. In a specific embodiment, the culture ismaintained at or about 36° C. In one embodiment, the temperature and/orpH of the culture is maintained for at least 7 days. In one embodiment,the method is performed is under continuous or fed-batch cultureconditions. In a specific embodiment, the continuous culture conditionis a chemostatic culture condition. In another specific embodiment, thecontinuous culture condition is a perfusion culture condition. In oneembodiment, the cell harboring the nucleic acid encoding an ADAMTS13protein is a mammalian cell. In particular embodiments, the mammaliancell is a hamster, human, or murine cell. In a specific embodiment, thecell is a CHO cell line, an HEK 293 cell line, or a BHK cell line. Inanother embodiment, the mammalian cell is cultured in an animal proteinand/or polypeptide free culture medium. In yet another embodiment, themammalian cell is cultured in a synthetic culture medium, which may ormay not be free of animal proteins and polypeptides. In one embodiment,the culture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about between 2 mg/L and 10 mg/L nicotinamide (vitamin B3)and at least at or about 0.5 mM calcium. In a related embodiment, theculture medium comprises at least at or about between 2 mg/L and 10 mg/Lnicotinamide (vitamin B3) and at least at or about 1.5 mM calcium. Inanother related embodiment, the culture medium comprises at least at orabout between 2 mg/L and 10 mg/L nicotinamide (vitamin B3) and at leastat or about between 0.5 mM and 1.5 mM calcium. In certain embodiments,the culture medium comprises at least at or about between 2 mg/L and 10mg/L nicotinamide (vitamin B3) and at least at or about 0.5 mM, or atleast at or about 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, or morecalcium. In one embodiment, the culture is maintained at or aboutbetween 35° C. and 37° C. In a specific embodiment, the culture ismaintained at or about 36° C. In one embodiment, the temperature and/orpH of the culture is maintained for at least 7 days. In one embodiment,the method is performed is under continuous or fed-batch cultureconditions. In a specific embodiment, the continuous culture conditionis a chemostatic culture condition. In another specific embodiment, thecontinuous culture condition is a perfusion culture condition. In oneembodiment, the cell harboring the nucleic acid encoding an ADAMTS13protein is a mammalian cell. In particular embodiments, the mammaliancell is a hamster, human, or murine cell. In a specific embodiment, thecell is a CHO cell line, an HEK 293 cell line, or a BHK cell line. Inanother embodiment, the mammalian cell is cultured in an animal proteinand/or polypeptide free culture medium. In yet another embodiment, themammalian cell is cultured in a synthetic culture medium, which may ormay not be free of animal proteins and polypeptides. In one embodiment,the culture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine.

In another embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising atleast at or about 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L, 7 mg/L, 8mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or higher concentrations ofnicotinamide (vitamin B3) and at least at or about 0.5 mM calcium. In arelated embodiment, the culture medium comprises at or about2 mg/L, 3mg/L, 4 mg/L, 5 mg/L, 6 mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L,20 mg/L, or higher concentrations of nicotinamide (vitamin B3) and atleast at or about 1.5 mM calcium. In another related embodiment, theculture medium comprises at or about 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or higherconcentrations of nicotinamide (vitamin B3) and at least at or aboutbetween 0.5 mM and 1.5 mM calcium. In certain embodiments, the culturemedium comprises at or about 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L, 7mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or higherconcentrations of nicotinamide (vitamin B3) and at least at or about 0.5mM, or at least at or about 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, ormore calcium. In one embodiment, the culture is maintained at or aboutbetween 35° C. and 37° C. In a specific embodiment, the culture ismaintained at or about 36° C. In one embodiment, the temperature and/orpH of the culture is maintained for at least 7 days. In one embodiment,the method is performed is under continuous or fed-batch cultureconditions. In a specific embodiment, the continuous culture conditionis a chemostatic culture condition. In another specific embodiment, thecontinuous culture condition is a perfusion culture condition. In oneembodiment, the cell harboring the nucleic acid encoding an ADAMTS13protein is a mammalian cell. In particular embodiments, the mammaliancell is a hamster, human, or murine cell. In a specific embodiment, thecell is a CHO cell line, an HEK 293 cell line, or a BHK cell line. Inanother embodiment, the mammalian cell is cultured in an animal proteinand/or polypeptide free culture medium. In yet another embodiment, themammalian cell is cultured in a synthetic culture medium, which may ormay not be free of animal proteins and polypeptides. In one embodiment,the culture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine.

In one embodiment, a method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs 13 (ADAMTS13) protein isprovided, the method comprising culturing a cell harboring a nucleicacid encoding an ADAMTS13 protein in a culture medium comprising zinc,calcium, and nicotinamide (vitamin B3). In certain embodiments, zinc ispresent in the culture medium at a concentration of at least at or about2 μM, 5 μM, between 2 μM and 12 μM, between 5 μM and 12 μM, or at least3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14μM, 15 μM, 20 μM, 25 μM, 30 μM, or more. In certain embodiments, calciumis present in the culture medium at a concentration of at least at orabout 0.5 mM, 1.5 mM, between 0.5 and 1.5, or at least 0.6 mM, 0.7 mM,0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM,1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5mM, 4.0 mM, 4.5 mM, 5.0 mM, or more. In certain embodiments,nicotinamide (vitamin B3) is present in the culture medium at aconcentration of at least at or about 2 mg/L, 7 mg/L, between 2 mg/L and7 mg/L, or at least at or about 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L, 7 mg/L,8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or more.

As expressly disclosed herein, and provided as Var. 14 to Var. 93 (Table3 to Table 6), any combination of concentrations of the three components(i.e., zinc, calcium, and nicotinamide (vitamin B3)) is contemplated foruse in the methods of the invention. In one embodiment, the culture ismaintained at or about between 35° C. and 37° C. In a specificembodiment, the culture is maintained at or about 36° C. In oneembodiment, the temperature and/or pH of the culture is maintained forat least 7 days. In one embodiment, the method is performed is undercontinuous or fed-batch culture conditions. In a specific embodiment,the continuous culture condition is a chemostatic culture condition. Inanother specific embodiment, the continuous culture condition is aperfusion culture condition. In one embodiment, the cell harboring thenucleic acid encoding an ADAMTS13 protein is a mammalian cell. Inparticular embodiments, the mammalian cell is a hamster, human, ormurine cell. In a specific embodiment, the cell is a CHO cell line, anHEK 293 cell line, or a BHK cell line. In another embodiment, themammalian cell is cultured in an animal protein and/or polypeptide freeculture medium. In yet another embodiment, the mammalian cell iscultured in a synthetic culture medium, which may or may not be free ofanimal proteins and polypeptides. In one embodiment, the culture mediumfurther comprises a polyamine at or about between 0.5 mg/L and 30 mg/L.In another embodiment, the culture medium further comprises a polyamineat or about between 2 mg/L and 8 mg/L. In a specific embodiment, thepolyamine is putrescine.

TABLE 3 Exemplary embodiments of culture mediums containing at least 2mg/L nicotinamide (vitamin B3), which are useful for the expression ofan ADAMTS13 protein. At least 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM,1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM,2.0 mM, Between 2.25 mM, 2.5 mM, 2.75 mM, At least At least 0.5 mM and3.0 mM, 3.5 mM, 4.0 mM, 0.5 mM 1.5 mM 1.5 mM 4.5 mM, 5.0 mM, or morecalcium calcium calcium calcium At least 2 μM Zinc Var. 14 Var. 15 Var.16 Var. 17 At least 5 μM Zinc Var. 18 Var. 19 Var. 20 Var. 21 Between 2μM and 12 μM Var. 22 Var. 23 Var. 24 Var. 25 zinc Between 5 μM and 12 μMVar. 26 Var. 27 Var. 28 Var. 29 zinc At least 3 μM, 4 μM, 5 μM, Var. 30Var. 31 Var. 32 Var. 33 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, or more zinc *Var. = Variation

TABLE 4 Exemplary embodiments of culture mediums containing at least 7mg/L nicotinamide (vitamin B3), which are useful for the expression ofan ADAMTS13 protein. At least 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM,1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM,2.0 mM, Between 2.25 mM, 2.5 mM, 2.75 mM, At least At least 0.5 mM and3.0 mM, 3.5 mM, 4.0 mM, 0.5 mM 1.5 mM 1.5 mM 4.5 mM, 5.0 mM, or morecalcium calcium calcium calcium At least 2 μM Zinc Var. 34 Var. 35 Var.36 Var. 37 At least 5 μM Zinc Var. 38 Var. 39 Var. 40 Var. 41 Between 2μM and 12 μM Var. 42 Var. 43 Var. 44 Var. 45 zinc Between 5 μM and 12 μMVar. 46 Var. 47 Var. 48 Var. 49 zinc At least 3 μM, 4 μM, 5 μM, Var. 50Var. 51 Var. 52 Var. 53 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, or more zinc *Var. = Variation

TABLE 5 Exemplary embodiments of culture mediums containing between 2and 7 mg/L nicotinamide (vitamin B3), which are useful for theexpression of an ADAMTS13 protein. At least 0.6 mM, 0.7 mM, 0.8 mM, 0.9mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8mM, 1.9 mM, 2.0 mM, Between 2.25 mM, 2.5 mM, 2.75 mM, At least At least0.5 mM and 3.0 mM, 3.5 mM, 4.0 mM, 0.5 mM 1.5 mM 1.5 mM 4.5 mM, 5.0 mM,or more calcium calcium calcium calcium At least 2 μM Zinc Var. 54 Var.55 Var. 56 Var. 57 At least 5 μM Zinc Var. 58 Var. 59 Var. 60 Var. 61Between 2 μM and 12 μM Var. 62 Var. 63 Var. 64 Var. 65 zinc Between 5 μMand 12 μM Var. 66 Var. 67 Var. 68 Var. 69 zinc At least 3 μM, 4 μM, 5μM, Var. 70 Var. 71 Var. 72 Var. 73 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, or more zinc *Var.= Variation

TABLE 6 Exemplary embodiments of culture mediums containing at least 3mg/L, 4 mg/L, 5 mg/L, 6 mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L,20 mg/L, or more nicotinamide (vitamin B3), which are useful for theexpression of an ADAMTS13 protein. At least 0.6 mM, 0.7 mM, 0.8 mM, 0.9mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8mM, 1.9 mM, 2.0 mM, Between 2.25 mM, 2.5 mM, 2.75 mM, At least At least0.5 mM and 3.0 mM, 3.5 mM, 4.0 mM, 0.5 mM 1.5 mM 1.5 mM 4.5 mM, 5.0 mM,or more calcium calcium calcium calcium At least 2 μM Zinc Var. 74 Var.75 Var. 76 Var. 77 At least 5 μM Zinc Var. 78 Var. 79 Var. 80 Var. 81Between 2 μM and 12 μM Var. 82 Var. 83 Var. 84 Var. 85 zinc Between 5 μMand 12 μM Var. 86 Var. 87 Var. 88 Var. 89 zinc At least 3 μM, 4 μM, 5μM, Var. 90 Var. 91 Var. 92 Var. 93 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, or more zinc *Var.= Variation

B. Host Cells and Vectors

Recombinant ADAMTS proteins can be produced by expression in anysuitable prokaryotic or eukaryotic host system. Examples of eukaryoticcells include, without limitation, mammalian cells, such as CHO, COS,HEK 293, BHK, SK-Hep, and HepG2; insect cells, for example SF9 cells,SF21 cells, S2 cells, and High Five cells; and yeast cells, for exampleSaccharomyces or Schizosaccharomyces cells. In one embodiment, theADAMTS proteins can be expressed in bacterial cells, yeast cells, insectcells, avian cells, mammalian cells, and the like. For example, in ahuman cell line, a hamster cell line, or a murine cell line. In oneparticular embodiment, the cell line is a CHO, BHK, or HEK cell line. Ina preferred embodiment, the cell line is a CHO cell line.

In one embodiment, the cells may be any mammalian cell that can becultured, preferably in a manufacturing process (i.e., at least 1liter), to produce a desired ADAMTS protein such as ADAMTS13. Examplesinclude the monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells/-DHFR, such as the DUKX-B11 subclone (CHO, Uriaub andChasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse Sertoli cells(TM4, Mather, Biol. Reprod, 23:243-251 (1980)); monkey kidney cells (CV1ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCCCRL-1587); human cervical carcinoma cells (HeLa, ATCC CCL 2); caninekidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells(Mather et al., Annals N. Y. Acad. Sci., 383:44-68 (1982)); MRC 5 cells;FS4 cells; and the human hepatoma line (Hep G2). Preferably, the cellline is a rodent cell line, especially a hamster cell line such as CHOor BHK.

A wide variety of vectors can be used for the expression of an ADAMTSprotein (e.g., ADAMTS13) and can be selected from eukaryotic andprokaryotic expression vectors. In certain embodiments, a plasmid vectoris contemplated for use in expressing an ADAMTS protein (e.g.,ADAMTS13). In general, plasmid vectors containing replicon and controlsequences which are derived from species compatible with the host cellare used in connection with these hosts. The vector can carry areplication site, as well as marking sequences which are capable ofproviding phenotypic selection in transformed cells. The plasmid willcomprise a nucleotide sequence encoding an ADAMTS protein (e.g.,ADAMTS13) operable linked to one or more control sequences, for example,a promoter.

A preferred method of preparing stable CHO cell clones expressing arecombinant ADAMTS protein is as follows. A DHFR deficient CHO cell lineDUKX-B 11 is transfected with a DHFR expression vector to allow forexpression of the relevant recombinant protein, essentially as describedin U.S. Pat. No. 5,250,421 (Kaufman et al., Genetics Institute, Inc.).Selection is carried out by growth in Hypoxanthine/Thymidine (HT) freemedia and amplification of the relevant region coding for expression ofthe recombinant ADAMTS protein and DHFR gene is achieved by propagationof the cells in increasing concentrations of methotrexate. Whereappropriate, CHO cell lines may be adapted for growth in serum and/orprotein free medium, essentially as described in U.S. Psy. No. 6,100,061(Reiter et al, lmmuno Aktiengesellschaft).

In another preferred embodiment, stable HEK293 cells are prepared bytransfecting with a construct containing a hygromycin selectable markerand selecting transformants by antibiotic resistance.

The ability of certain viruses to infect cells or enter cells viareceptor-mediated endocytosis, and to integrate into host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g.,mammalian cells). Accordingly, in certain embodiments, a viral vector isused to introduce a nucleotide sequence encoding an ADAMTS protein(e.g., ADAMTS13) into a host cell for expression. The viral vector willcomprise a nucleotide sequence encoding an ADAMTS protein (e.g.,ADAMTS13) operable linked to one or more control sequences, for example,a promoter. Alternatively, the viral vector may not contain a controlsequence and will instead rely on a control sequence within the hostcell to drive expression of the ADAMTS protein. Non-limiting examples ofvirus vectors that may be used to deliver a nucleic acid includeAdenoviral vectors, AAV vectors, and Retroviral vectors.

In one embodiment, an Adenovirus expression vector include thoseconstructs containing adenovirus sequences sufficient to supportpackaging of the construct and to ultimately express an ADAMTS constructthat has been cloned therein. Adenoviral vectors allow for theintroduction of foreign sequences up to 7 kb (Grunhaus et al., Seminarin Virology, 200(2):535-546, 1992)).

In another embodiment, an adeno-associated virus (AAV) can be used tointroduce a nucleotide sequence encoding an ADAMTS protein (e.g.,ADAMTS13) into a host cell for expression. AAV systems have beendescribed previously and are generally well known in the art (Kelleherand Vos, Biotechniques, 17(6):1110-7, 1994; Cotten et al., Proc NatlAcad Sci USA, 89(13):6094-6098, 1992; Curiel, Nat Immun, 13(2-3):141-64,1994; Muzyczka, Curr Top Microbiol Immunol, 158:97-129, 1992). Detailsconcerning the generation and use of rAAV vectors are described, forexample, in U.S. Patent Nos. 5,139,941 and 4,797,368, each incorporatedherein by reference in their entireties for all purposes.

In one embodiment, a retroviral expression vector can be used tointroduce a nucleotide sequence encoding an ADAMTS protein (e.g.,ADAMTS13) into a host cell for expression. These systems have beendescribed previously and are generally well known in the art (Mann etal., Cell, 33:153-159, 1983; Nicolas and Rubinstein, In: Vectors: Asurvey of molecular cloning vectors and their uses, Rodriguez andDenhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988; Temin, In:Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188,1986). In a specific embodiment, the retroviral vector is a lentiviralvector (see, for example, Naldini et al., Science, 272(5259):263-267,1996; Zufferey et al., Nat Biotechnol, 15(9):871-875, 1997; Blomer etal., J Virol., 71(9):6641-6649, 1997; U.S. Pat. Nos. 6,013,516 and5,994,136).

Non-limiting examples of vectors for prokaryotic expression includeplasmids such as pRSET, pET, pBAD, etc., wherein the promoters used inprokaryotic expression vectors include lac, trc, trp, recA, araBAD, etc.Examples of vectors for eukaryotic expression include: (i) forexpression in yeast, vectors such as pAO, pPIC, pYES, pMET, usingpromoters such as AOX1, GAP, GAL1, AUG1, etc; (ii) for expression ininsect cells, vectors such as pMT, pAc5, pIB, pMIB, pBAC, etc., usingpromoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh, etc., and (iii)for expression in mammalian cells, vectors such as pSVL, pCMV, pRc/RSV,pcDNA3, pBPV, etc., and vectors derived form viral systems such asvaccinia virus, adeno-associated viruses, herpes viruses, retroviruses,etc., using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, andβ-actin.

In certain embodiments, the cell-culture methods of the invention maycomprise the use of a microcarrier. The present invention provides,among other aspect, methods of large-scale ADAMTS protein expression. Insome embodiments, the cell-cultures of the embodiments can be performedin large bioreactors under conditions suitable for providing highvolume-specific culture surface areas to achieve high cell densities andprotein expression. One means for providing such growth conditions is touse microcarriers for cell-culture in stirred tank bioreactors. Inanother embodiment, these growth requirements are met via the use of asuspension cell culture.

C. Cultivation Methods

In certain embodiments, the methods of the present invention cancomprise the use of a cell culture system operated under a batch orcontinuous mode of operation. For example, when batch cell cultures areutilized, they may be operated under single batch, fed-batch, orrepeated-batch mode. Likewise, continuous cell cultures may be operatedunder, for example, perfusion, turbidostat or chemostat mode. Batch andcontinuous cell cultivation may be performed under either suspension oradherence conditions. When operated under suspension conditions, thecells will be freely suspended and mixed within the culture medium.Alternatively, under adherence conditions, the cells will be bound to asolid phase, for example, a microcarrier, a porous microcarrier, diskcarrier, ceramic cartridge, hollow fiber, flat sheet, gel matrix, andthe like.

A batch culture is typically a large scale cell culture in which a cellinoculum is cultured to a maximum density in a tank or fermenter, andharvested and processed as a single batch. A fed-batch culture ittypically a batch culture which is supplied with either fresh nutrients(e.g., growth-limiting substrates) or additives (e.g., precursors toproducts). The feed solution is usually highly concentrated to avoiddilution of the bioreactor. In a repeated-batch culture, the cells areplaced in a culture medium and grown to a desired cell density. To avoidthe onset of a decline phase and cell death, the culture is then dilutedwith complete growth medium before the cells reach their maximumconcentration. The amount and frequency of dilution varies widely anddepends on the growth characteristics of the cell line and convenienceof the culture process. The process can be repeated as many times asrequired and, unless cells and medium are discarded at subculture, thevolume of culture will increase stepwise as each dilution is made. Theincreasing volume may be handled by having a reactor of sufficient sizeto allow dilutions within the vessel or by dividing the diluted cultureinto several vessels. The rationale of this type of culture is tomaintain the cells in an exponentially growing state. Serial subcultureis characterized in that the volume of culture is always increasingstepwise, there can be multiple harvests, the cells continue to grow andthe process can continue for as long as desired. In certain embodiments,an ADAMTS protein (e.g., ADAMTS13) may be recovered after harvesting thesupernatant of a batch culture.

A continuous culture can be a suspension culture that is continuouslysupplied with nutrients by the inflow of fresh medium, wherein theculture volume is usually kept constant by the concomitant removal ofspent medium. In chemostat and turbidostat methods, the extracted mediumcontains cells. Thus, the cells remaining in the cell culture vesselmust grow to maintain a steady state. In the chemostat method, thegrowth rate is typically controlled by controlling the dilution rate,i.e., the rate at which fresh medium is added. The growth rate of thecells in the culture may be controlled, for example, at a sub-maximalgrowth rate, by alteration of the dilution rate. In contrast, in theturbidostat method, the dilution rate is set to permit the maximumgrowth rate that the cells can achieve at the given operatingconditions, such as pH and temperature.

In a perfusion culture, the extracted medium is depleted of cells, whichare retained in the culture vessel, for example, by filtration or bycentrifugal methods that lead to the reintroduction of the cells intothe culture. However, typically membranes used for filtration do notretain 100% of cells, and so a proportion are removed when the medium isextracted. It may not be crucial to operate perfusion cultures at veryhigh growth rates, as the majority of the cells are retained in theculture vessel.

Stirred-tank reactor system can be used for batch and continuous cellcultures operated under suspension or adherent modes. Generally, thestirred-tank reactor system can be operated as any conventionalstirred-tank reactor with any type of agitator such as a Rushton,hydrofoil, pitched blade, or marine.

The methods of the invention may comprise the use of fed-batchcell-cultivation or continuous cell-cultivation, such as perfusion orchemostatic cell-cultivation, for the expression of an ADAMTS protein.In certain embodiments, it was found that fed-batch culture mediumssupplemented with zinc at concentrations up to at least about 12 μMprovided for expression of an ADAMTS protein with increased specificactivities (Table 20). Notably, in fed-batch cultures supplemented withzinc at a final concentration of 12 μM, specific cell growth rates andtotal activity were unaffected, while the specific activities of theexpressed ADAMTS13 proteins continued to increase. This is in contrastto what was seen in experiments employing chemostatic cell-cultivation.In these experiments, supplementation of the culture medium with zinc ata final concentration of 5 μM resulted in an increase in the specificactivity of the ADAMTS13 in the supernatant (Table 21). However, athigher levels of supplementation, 8.5 μM and 12 μM, specific cell growthrates and total ADAMTS13 protein yields were decreased, although thespecific activity of ADAMTS13 in the culture supernatant remained high.

Accordingly, in one embodiment the methods of the invention comprise theuse of fed-batch cell-cultivation with a medium comprising zinc at aconcentration of at least at or about 2 μM, at least at or about 5 μM,at or about between 2 μM and 12 μM, at or about between 2 μM and 5 μM,at or about between 5 μM and 12 μM, or at least at or about 3 μM, 4 μM,5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM,20 μM, 25 μM, 30 μM, or more zinc. In some embodiments, the zincconcentration may be from at least about 5 μM to at least about 12 μM.In certain embodiments, the medium may be an animal protein-free,oligopeptide-free, or chemically defined medium.

1. Continuous Culture

Advantageously, the present invention provides methods of expressing anADAMTS protein, for example ADAMTS13, with high specific activity in acontinuous culture. These methods allow for the continued expression andpurification of an ADAMTS proteins from a single culture over extendedperiods of time. Specifically, it was found that high levels of ADAMTS13protein expression and specific activity could be maintained for atleast 53 days in a 10 L chemostat bioreactor, under the conditionsprovided by the present invention (Example 3, see, Table 15). In apreferred embodiment, the ADAMTS protein is ADAMTS13.

Accordingly, in one embodiment, the present invention provides methodsof expressing an ADAMTS protein (e.g., ADAMTS13) with high specificityfor an extended period of time. In certain embodiments, the culture ismaintained for at least at or about 7 days, or at least at or about 14days, 21 days, 28 days, or at least at or about 5 weeks, 6 weeks, 7weeks, or at least at or about 2 months, or 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18 months or longer. In some of theseembodiments, the level of total ADAMTS protein expression, activity, orspecific activity is maintained in the culture for an extended period oftime. In other embodiments, the specific growth rate, cell density, andthe like is maintained in the culture for an extended period of time. Insome embodiments, the culture medium may be supplemented with at leastone of calcium, zinc, or nicotinamide (vitamin B3), for example, at aconcentration according to any one of Var. 1 to Var. 149 (Table 2 toTable 9). In one particular embodiment, the method comprises the use ofchemostat cell-cultivation. In another embodiment, the method comprisesthe use of turbidostat cell-cultivation. In yet another embodiment, themethod comprises the use of perfusion cell-cultivation. In certainembodiments, the medium may be an animal protein-free,oligopeptide-free, or chemically defined medium. In a preferredembodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, a continuous cell cultivation technique may be usedto express an ADAMTS protein (e.g., ADAMTS13) in a cell culturecontaining zinc, calcium, and/or nicotinamide (vitamin B3) at aconcentration according to any one of Var. 1 to Var. 149 (Table 2 toTable 9) for at least 7 days. In another embodiment, a continuous cellcultivation technique may be used to express an ADAMTS protein (e.g.,ADAMTS13) in a cell culture containing zinc, calcium, and/ornicotinamide (vitamin B3) at a concentration according to any one ofVar. 1 to Var. 149 (Table 2 to Table 9) for at least 14 days. In anotherembodiment, a continuous cell cultivation technique may be used toexpress an ADAMTS protein (e.g., ADAMTS13) in a cell culture containingzinc, calcium, and/or nicotinamide (vitamin B3) at a concentrationaccording to any one of Var. 1 to Var. 149 (Table 2 to Table 9) for atleast 21 days. In another embodiment, a continuous cell cultivationtechnique may be used to express an ADAMTS protein (e.g., ADAMTS13) in acell culture containing zinc, calcium, and/or nicotinamide (vitamin B3)at a concentration according to any one of Var. 1 to Var. 149 (Table 2to Table 9) for at least 1 month. In another embodiment, a continuouscell cultivation technique may be used to express an ADAMTS protein(e.g., ADAMTS13) in a cell culture containing zinc, calcium, and/ornicotinamide (vitamin B3) at a concentration according to any one ofVar. 1 to Var. 149 (Table 2 to Table 9) for at least 2 months. Inanother embodiment, a continuous cell cultivation technique may be usedto express an ADAMTS protein (e.g., ADAMTS13) in a cell culturecontaining zinc, calcium, and/or nicotinamide (vitamin B3) at aconcentration according to any one of Var. 1 to Var. 149 (Table 2 toTable 9) for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, or more months. In a preferred embodiment, the ADAMTS protein is

In certain embodiments, the methods of ADAMTS protein (e.g., ADAMTS13)expression may comprise the use of continuous cell-cultivation with amedium comprising zinc at a concentration at of at least at or about 2μM zinc, at least at or about 5 μM zinc, at or about between 2 μM and 12μM zinc, at or about between 2 μM and 5 μM zinc, at or about between 3μM and 5 μM zinc, at or about between 5 μM and 12 μM zinc, or at leastat or about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, or more zinc for at least7 days. In one particular embodiment, the method comprises the use ofchemostat cell-cultivation. In another embodiment, the method comprisesthe use of turbidostat cell-cultivation. In yet another embodiment, themethod comprises the use of perfusion cell-cultivation. In certainembodiments, the medium may be an animal protein-free,oligopeptide-free, or chemically defined medium. In a preferredembodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, a method for the expressing an ADAMTS proteincomprises culturing, under continuous culture conditions, a cellharboring a nucleic acid encoding an ADAMTS protein (e.g., ADAMTS13) ina culture medium containing at least at or about 2 μM zinc for at least7 days. In other embodiments, the culture medium will contain at leastabout 3 μM zinc. In another embodiment, the culture medium will containat least about 5 μM zinc. In another embodiment, the culture medium willcontain at or about between 2 μM zinc and 5 μM zinc. In anotherembodiment, the culture medium will contain at or about between 3 μMzinc and 5 μM zinc. In another embodiment, the culture medium willcontain at or about between 2 μM zinc and 12 μM zinc. In anotherembodiment, the culture medium will contain at or about between 3 μMzinc and 12 μM zinc. In another embodiment, the culture medium willcontain at or about between 5 μM zinc and 12 μM zinc. In anotherembodiment, the culture medium will contain at least at or about 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15μM, 20 μM, 25 μM, 30 μM, or more zinc. In certain embodiments, theculture is maintained for at least 7 days, or at least 14 days, 21 days,28 days, or at least 5 weeks, 6 weeks, 7 weeks, or at least 2 months, or3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 months orlonger. In one particular embodiment, the method comprises the use ofchemostat cell-cultivation. In another embodiment, the method comprisesthe use of turbidostat cell-cultivation. In yet another embodiment, themethod comprises the use of perfusion cell-cultivation. In certainembodiments, the medium may be an animal protein-free,oligopeptide-free, or chemically defined medium. In a preferredembodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, a method for the expressing an ADAMTS proteincomprises culturing, under continuous culture conditions, a cellharboring a nucleic acid encoding an ADAMTS protein (e.g., ADAMTS13) ina culture medium containing at least at or about 0.5 mM calcium for atleast 7 days. In other embodiments, the culture medium will contain atleast about 1.0 mM calcium. In another embodiment, the culture mediumwill contain at least about 1.5 mM calcium. In another embodiment, theculture medium will contain at or about between 0.5 mM and 1.5 mMcalcium. In another embodiment, the culture medium will contain at orabout between 1.0 mM and 1.5 mM calcium. In another embodiment, theculture medium will contain at least at or about 0.6 mM, 0.7 mM, 0.8 mM,0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM,1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5 mM, 4.0mM, 4.5 mM, 5.0 mM, or more calcium. In certain embodiments, the cultureis maintained for at least 7 days, or at least 14 days, 21 days, 28days, or at least 5 weeks, 6 weeks, 7 weeks, or at least 2 months, or 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 months or longer.In one particular embodiment, the method comprises the use of chemostatcell-cultivation. In another embodiment, the method comprises the use ofturbidostat cell-cultivation. In yet another embodiment, the methodcomprises the use of perfusion cell-cultivation. In certain embodiments,the medium may be an animal protein-free, oligopeptide-free, orchemically defined medium. In a preferred embodiment, the ADAMTS proteinis ADAMTS13.

In one embodiment, a method for the expressing an ADAMTS proteincomprises culturing, under continuous culture conditions, a cellharboring a nucleic acid encoding an ADAMTS protein (e.g., ADAMTS13) ina culture medium containing at least at or about 2 mg/L nicotinamide(vitamin B3) for at least 7 days. In other embodiments, the culturemedium will contain at least about 5 mg/L nicotinamide (vitamin B3). Inanother embodiment, the culture medium will contain at least about 7mg/L nicotinamide (vitamin B3). In another embodiment, the culturemedium will contain at or about between 2 mg/L and 7 mg/L nicotinamide(vitamin B3). In another embodiment, the culture medium will contain ator about between 5 mg/L and 7 mg/L nicotinamide (vitamin B3). In anotherembodiment, the culture medium will contain at least at or about 3 mg/L,4 mg/L, 5 mg/L, 6 mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20mg/L, or more nicotinamide (vitamin B3). In certain embodiments, theculture is maintained for at least 7 days, or at least 14 days, 21 days,28 days, or at least 5 weeks, 6 weeks, 7 weeks, or at least 2 months, or3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 months orlonger. In one particular embodiment, the method comprises the use ofchemostat cell-cultivation. In another embodiment, the method comprisesthe use of turbidostat cell-cultivation. In yet another embodiment, themethod comprises the use of perfusion cell-cultivation. In certainembodiments, the medium may be an animal protein-free,oligopeptide-free, or chemically defined medium. In a preferredembodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, a method for the expressing an ADAMTS proteincomprises culturing, under continuous culture conditions, a cellharboring a nucleic acid encoding an ADAMTS protein (e.g., ADAMTS13) ina culture medium supplemented with both zinc and calcium for at least 7days. In certain embodiments, the zinc and calcium concentrations can beany of those described herein. In certain embodiments, the zinc andcalcium concentrations will be one of Var. 94 to Var. 113 (Table 7). Incertain embodiments, the culture is maintained for at least 7 days, orat least 14 days, 21 days, 28 days, or at least 5 weeks, 6 weeks, 7weeks, or at least 2 months, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18 months or longer. In one particular embodiment, themethod comprises the use of chemostat cell-cultivation. In anotherembodiment, the method comprises the use of turbidostatcell-cultivation. In yet another embodiment, the method comprises theuse of perfusion cell-cultivation. In certain embodiments, the mediummay be an animal protein-free, oligopeptide-free, or chemically definedmedium. In a preferred embodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, a method for the expressing an ADAMTS proteincomprises culturing, under continuous culture conditions, a cellharboring a nucleic acid encoding an ADAMTS protein (e.g., ADAMTS13) ina culture medium supplemented with both zinc and nicotinamide (vitaminB3) for at least 7 days. In certain embodiments, the zinc andnicotinamide (vitamin B3) concentrations can be any of those describedherein. In certain embodiments, the zinc and nicotinamide (vitamin B3)concentrations will be one of Var. 114 to Var. 133 (Table 8). In certainembodiments, the culture is maintained for at least 7 days, or at least14 days, 21 days, 28 days, or at least 5 weeks, 6 weeks, 7 weeks, or atleast 2 months, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18 months or longer. In one particular embodiment, the method comprisesthe use of chemostat cell-cultivation. In another embodiment, the methodcomprises the use of turbidostat cell-cultivation. In yet anotherembodiment, the method comprises the use of perfusion cell-cultivation.In certain embodiments, the medium may be an animal protein-free,oligopeptide-free, or chemically defined medium. In a preferredembodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, a method for the expressing an ADAMTS proteincomprises culturing, under continuous culture conditions, a cellharboring a nucleic acid encoding an ADAMTS protein (e.g., ADAMTS13) ina culture medium supplemented with both calcium and nicotinamide(vitamin B3) for at least 7 days. In certain embodiments, the calciumand nicotinamide (vitamin B3) concentrations can be any of thosedescribed herein. In certain embodiments, the calcium and nicotinamide(vitamin B3) concentrations will be one of Var. 134 to Var. 149 (Table9). In certain embodiments, the culture is maintained for at least 7days, or at least 14 days, 21 days, 28 days, or at least 5 weeks, 6weeks, 7 weeks, or at least 2 months, or 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18 months or longer. In one particularembodiment, the method comprises the use of chemostat cell-cultivation.In another embodiment, the method comprises the use of turbidostatcell-cultivation. In yet another embodiment, the method comprises theuse of perfusion cell-cultivation. In certain embodiments, the mediummay be an animal protein-free, oligopeptide-free, or chemically definedmedium. In a preferred embodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, a method for the expressing an ADAMTS proteincomprises culturing, under continuous culture conditions, a cellharboring a nucleic acid encoding an ADAMTS protein (e.g., ADAMTS13) ina culture medium supplemented with zinc, calcium, and nicotinamide(vitamin B3) for at least 7 days. In certain embodiments, the zinc,calcium, and nicotinamide (vitamin B3) concentrations can be any ofthose described herein. In certain embodiments, the zinc, calcium, andnicotinamide (vitamin B3) concentrations will be one of Var. 14 to Var.93 (Table 3 to Table 6). In certain embodiments, the culture ismaintained for at least 7 days, or at least 14 days, 21 days, 28 days,or at least 5 weeks, 6 weeks, 7 weeks, or at least 2 months, or 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 months or longer. In oneparticular embodiment, the method comprises the use of chemostatcell-cultivation. In another embodiment, the method comprises the use ofturbidostat cell-cultivation. In yet another embodiment, the methodcomprises the use of perfusion cell-cultivation. In certain embodiments,the medium may be an animal protein-free, oligopeptide-free, orchemically defined medium. In a preferred embodiment, the ADAMTS proteinis ADAMTS13.

In one embodiment, the culture is maintained at a cell density ofbetween about 0.5×10⁶ and 4×10⁷ cells/ml for an extended period of time.In other embodiments, the cell density is maintained at a concentrationof between about 1.0×10⁶ and about 1.0×10⁷ cells/ml for an extendedperiod of time. In other embodiments, the cell density is maintained ata concentration of between about 1.0×10⁶ and about 4.0×10⁶ cells/ml foran extended period of time. In other embodiments, the cell density ismaintained at a concentration of between about 1.0×10⁶ and about 4.0×10⁶cells/ml for an extended period of time. In yet other embodiments, thecell density may be maintained at a concentration between about 2.0×10⁶and about 4.0×10⁶, or between about 1.0×10⁶ and about 2.5×10⁶, orbetween about 1.5×10⁶ and about 3.5×10⁶, or any other similar range, foran extended period of time. The cell density at which a cell-culture ismaintained at for production of a recombinant ADAMTS protein (e.g.,ADAMTS13) will depend upon the culture-conditions and medium used forprotein expression. One of skill in the art will readily be able todetermine the optimal cell density for a cell-culture producing anADAMTS protein. In certain embodiments, the culture is maintained for atleast 7 days, or at least 14 days, 21 days, 28 days, or at least 5weeks, 6 weeks, 7 weeks, or at least 2 months, or 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18 months or longer. In one particularembodiment, the method comprises the use of chemostat cell-cultivation.In another embodiment, the method comprises the use of turbidostatcell-cultivation. In yet another embodiment, the method comprises theuse of perfusion cell-cultivation. In certain embodiments, the mediummay be an animal protein-free, oligopeptide-free, or chemically definedmedium. In a preferred embodiment, the ADAMTS protein is ADAMTS13.

In other embodiments, the methods of ADAMTS13 expression allow for theproduction of at least at or about 500 units of ADAMTS13 activity perliter culture per day (500 U/L/D), for example FRETS-VWF73 activityunits, with a specific activity of at least at or about 500 U/mg A13. Inanother embodiments, the methods of ADAMTS13 expression allow for theproduction of at least at or about 600 U/L/D. In another embodiments,the methods of ADAMTS13 expression allow for the production of at leastat or about 700 U/L/D. In another embodiments, the methods of ADAMTS13expression allow for the production of at least at or about 800 U/L/D.In another embodiments, the methods of ADAMTS13 expression allow for theproduction of at least at or about 900 U/L/D. In another embodiments,the methods of ADAMTS13 expression allow for the production of at leastat or about 1000 U/L/D. In another embodiments, the methods of ADAMTS13expression allow for the production of at least at or about 1100 U/L/D.In another embodiments, the methods of ADAMTS13 expression allow for theproduction of at least at or about 1200 U/L/D. In another embodiments,the methods of ADAMTS13 expression allow for the production of at leastat or about 1300 U/L/D. In another embodiments, the methods of ADAMTS13expression allow for the production of at least at or about 1400 U/L/D.In another embodiments, the methods of ADAMTS13 expression allow for theproduction of at least at or about 1500 U/L/D. In yet anotherembodiments, the methods of ADAMTS13 expression allow for the productionof at least at or about 2000 U/L/D. In one particular embodiment, themethod comprises the use of chemostat cell-cultivation. In anotherembodiment, the method comprises the use of turbidostatcell-cultivation. In yet another embodiment, the method comprises theuse of perfusion cell-cultivation. In certain embodiments, the mediummay be an animal protein-free, oligopeptide-free, or chemically definedmedium. In a preferred embodiment, the ADAMTS protein is ADAMTS13.

In another embodiment, the methods allow for the extended expression ofADAMTS13 protein with high specific activities, for example, a specificactivity of at least about 600U/mg A13 protein, or at least about 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, or more U/mg A13 protein for an extended period of time. Incertain embodiments, the culture is maintained for at least 7 days, orat least 14 days, 21 days, 28 days, or at least 5 weeks, 6 weeks, 7weeks, or at least 2 months, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18 months or longer. In one particular embodiment, themethod comprises the use of chemostat cell-cultivation. In anotherembodiment, the method comprises the use of turbidostatcell-cultivation. In yet another embodiment, the method comprises theuse of perfusion cell-cultivation. In certain embodiments, the mediummay be an animal protein-free, oligopeptide-free, or chemically definedmedium. In a preferred embodiment, the ADAMTS protein is ADAMTS13.

2. Batch Culture

In another aspect, the present invention provides methods of expressingan ADAMTS protein, for example ADAMTS13, with high specific activity ina batch culture. In some embodiments, the culture medium may besupplemented with at least one of calcium, zinc, or nicotinamide(vitamin B3), for example, at a concentration according to any one ofVar. 1 to Var. 149 (Table 2 to Table 9). In one particular embodiment,the method comprises the use of single-batch cell-cultivation. Inanother embodiment, the method comprises the use of fed-batchcell-cultivation. In yet another embodiment, the method comprises theuse of repeated batch cell-cultivation. In some embodiments, thecultures may be performed as suspension batch cultures. In otherembodiments, the cultures may be performed as adherent batch cultures.In certain embodiments, the medium may be an animal protein-free,oligopeptide-free, or chemically defined medium. In a preferredembodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, a fed-batch or repeated-batch cell cultivationtechnique may be used to express an ADAMTS protein (e.g., ADAMTS13) in acell culture containing zinc, calcium, and/or nicotinamide (vitamin B3)at a concentration according to any one of Var. 1 to Var. 149 (Table 2to Table 9) for at least 7 days. In another embodiment, a fed-batch orrepeated-batch cell cultivation technique may be used to express anADAMTS protein (e.g., ADAMTS13) in a cell culture containing zinc,calcium, and/or nicotinamide (vitamin B3) at a concentration accordingto any one of Var. 1 to Var. 149 (Table 2 to Table 9) for at least 14days. In another embodiment, a fed-batch or repeated-batch cellcultivation technique may be used to express an ADAMTS protein (e.g.,ADAMTS13) in a cell culture containing zinc, calcium, and/ornicotinamide (vitamin B3) at a concentration according to any one ofVar. 1 to Var. 149 (Table 2 to Table 9) for at least 21 days. In anotherembodiment, a fed-batch or repeated-batch cell cultivation technique maybe used to express an ADAMTS protein (e.g., ADAMTS13) in a cell culturecontaining zinc, calcium, and/or nicotinamide (vitamin B3) at aconcentration according to any one of Var. 1 to Var. 149 (Table 2 toTable 9) for at least 1 month. In another embodiment, a fed-batch orrepeated-batch cell cultivation technique may be used to express anADAMTS protein (e.g., ADAMTS13) in a cell culture containing zinc,calcium, and/or nicotinamide (vitamin B3) at a concentration accordingto any one of Var. 1 to Var. 149 (Table 2 to Table 9) for at least 2months. In another embodiment, a fed-batch or repeated-batch cellcultivation technique may be used to express an ADAMTS protein (e.g.,ADAMTS13) in a cell culture containing zinc, calcium, and/ornicotinamide (vitamin B3) at a concentration according to any one ofVar. 1 to Var. 149 (Table 2 to Table 9) for at least 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more months. In someembodiments, the cultures may be performed as suspension batch cultures.In other embodiments, the cultures may be performed as adherent batchcultures. In certain embodiments, the medium may be an animalprotein-free, oligopeptide-free, or chemically defined medium. In apreferred embodiment, the ADAMTS protein is ADAMTS13.

In certain embodiments, the methods of ADAMTS protein (e.g., ADAMTS13)expression may comprise the use of fed-batch or repeated-batchcell-cultivation with a medium comprising zinc at a concentration at ofat least at or about 2 μM zinc, at least at or about 5 μM zinc, at orabout between 2 μM and 12 μM zinc, at or about between 2 μM and 5 μMzinc, at or about between 3 μM and 5 μM zinc, at or about between 5 μMand 12 μM zinc, or at least at or about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM,or more zinc for at least 7 days. In some embodiments, the cultures maybe performed as suspension batch cultures. In other embodiments, thecultures may be performed as adherent batch cultures. In certainembodiments, the medium may be an animal protein-free,oligopeptide-free, or chemically defined medium. In a preferredembodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, a method for the expressing an ADAMTS proteincomprises culturing, under fed-batch or repeated-batch cultureconditions, a cell harboring a nucleic acid encoding an ADAMTS protein(e.g., ADAMTS13) in a culture medium containing at least at or about 0.5mM calcium for at least 7 days. In other embodiments, the culture mediumwill contain at least about 1.0 mM calcium. In another embodiment, theculture medium will contain at least about 1.5 mM calcium. In anotherembodiment, the culture medium will contain at or about between 0.5 mMand 1.5 mM calcium. In another embodiment, the culture medium willcontain at or about between 1.0 mM and 1.5 mM calcium. In anotherembodiment, the culture medium will contain at least at or about 0.6 mM,0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM,1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, or more calcium. In certainembodiments, the culture is maintained for at least 7 days, or at least14 days, 21 days, 28 days, or at least 5 weeks, 6 weeks, 7 weeks, or atleast 2 months, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18 months or longer. In some embodiments, the cultures may be performedas suspension batch cultures. In other embodiments, the cultures may beperformed as adherent batch cultures. In certain embodiments, the mediummay be an animal protein-free, oligopeptide-free, or chemically definedmedium. In a preferred embodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, a method for the expressing an ADAMTS proteincomprises culturing, under fed-batch or repeated-batch cultureconditions, a cell harboring a nucleic acid encoding an ADAMTS protein(e.g., ADAMTS13) in a culture medium containing at least at or about 2mg/L nicotinamide (vitamin B3) for at least 7 days. In otherembodiments, the culture medium will contain at least about 5 mg/Lnicotinamide (vitamin B3). In another embodiment, the culture mediumwill contain at least about 7 mg/L nicotinamide (vitamin B3). In anotherembodiment, the culture medium will contain at or about between 2 mg/Land 7 mg/L nicotinamide (vitamin B3). In another embodiment, the culturemedium will contain at or about between 5 mg/L and 7 mg/L nicotinamide(vitamin B3). In another embodiment, the culture medium will contain atleast at or about 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L, 7 mg/L, 8 mg/L, 9mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or more nicotinamide (vitamin B3). Incertain embodiments, the culture is maintained for at least 7 days, orat least 14 days, 21 days, 28 days, or at least 5 weeks, 6 weeks, 7weeks, or at least 2 months, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18 months or longer. In some embodiments, the cultures maybe performed as suspension batch cultures. In other embodiments, thecultures may be performed as adherent batch cultures. In certainembodiments, the medium may be an animal protein-free,oligopeptide-free, or chemically defined medium. In a preferredembodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, a method for the expressing an ADAMTS proteincomprises culturing, under fed-batch or repeated-batch cultureconditions, a cell harboring a nucleic acid encoding an ADAMTS protein(e.g., ADAMTS13) in a culture medium supplemented with both zinc andcalcium for at least 7 days. In certain embodiments, the zinc andcalcium concentrations can be any of those described herein. In certainembodiments, the zinc and calcium concentrations will be one of Var. 94to Var. 113 (Table 7). In certain embodiments, the culture is maintainedfor at least 7 days, or at least 14 days, 21 days, 28 days, or at least5 weeks, 6 weeks, 7 weeks, or at least 2 months, or 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18 months or longer. In someembodiments, the cultures may be performed as suspension batch cultures.In other embodiments, the cultures may be performed as adherent batchcultures. In certain embodiments, the medium may be an animalprotein-free, oligopeptide-free, or chemically defined medium. In apreferred embodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, a method for the expressing an ADAMTS proteincomprises culturing, under fed-batch or repeated-batch cultureconditions, a cell harboring a nucleic acid encoding an ADAMTS protein(e.g., ADAMTS13) in a culture medium supplemented with both zinc andnicotinamide (vitamin B3) for at least 7 days. In certain embodiments,the zinc and nicotinamide (vitamin B3) concentrations can be any ofthose described herein. In certain embodiments, the zinc andnicotinamide (vitamin B3) concentrations will be one of Var. 114 to Var.133 (Table 8). In certain embodiments, the culture is maintained for atleast 7 days, or at least 14 days, 21 days, 28 days, or at least 5weeks, 6 weeks, 7 weeks, or at least 2 months, or 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18 months or longer. In someembodiments, the cultures may be performed as suspension batch cultures.In other embodiments, the cultures may be performed as adherent batchcultures. In certain embodiments, the medium may be an animalprotein-free, oligopeptide-free, or chemically defined medium. In apreferred embodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, a method for the expressing an ADAMTS proteincomprises culturing, under fed-batch or repeated-batch cultureconditions, a cell harboring a nucleic acid encoding an ADAMTS protein(e.g., ADAMTS13) in a culture medium supplemented with both calcium andnicotinamide (vitamin B3) for at least 7 days. In certain embodiments,the calcium and nicotinamide (vitamin B3) concentrations can be any ofthose described herein. In certain embodiments, the calcium andnicotinamide (vitamin B3) concentrations will be one of Var. 134 to Var.149 (Table 9). In certain embodiments, the culture is maintained for atleast 7 days, or at least 14 days, 21 days, 28 days, or at least 5weeks, 6 weeks, 7 weeks, or at least 2 months, or 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18 months or longer. In someembodiments, the cultures may be performed as suspension batch cultures.In other embodiments, the cultures may be performed as adherent batchcultures. In certain embodiments, the medium may be an animalprotein-free, oligopeptide-free, or chemically defined medium. In apreferred embodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, a method for the expressing an ADAMTS proteincomprises culturing, under fed-batch or repeated-batch cultureconditions, a cell harboring a nucleic acid encoding an ADAMTS protein(e.g., ADAMTS13) in a culture medium supplemented with zinc, calcium,and nicotinamide (vitamin B3) for at least 7 days. In certainembodiments, the zinc, calcium, and nicotinamide (vitamin B3)concentrations can be any of those described herein. In certainembodiments, the zinc, calcium, and nicotinamide (vitamin B3)concentrations will be one of Var. 14 to Var. 93 (Table 3 to Table 6).In certain embodiments, the culture is maintained for at least 7 days,or at least 14 days, 21 days, 28 days, or at least 5 weeks, 6 weeks, 7weeks, or at least 2 months, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18 months or longer. In some embodiments, the cultures maybe performed as suspension batch cultures. In other embodiments, thecultures may be performed as adherent batch cultures. In certainembodiments, the medium may be an animal protein-free,oligopeptide-free, or chemically defined medium. In a preferredembodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, the culture is maintained at a cell density ofbetween about 0.5×10⁶ and 4×10⁷ cells/ml for an extended period of time.In other embodiments, the cell density is maintained at a concentrationof between about 1.0×10⁶ and about 1.0×10⁷ cells/ml for an extendedperiod of time. In other embodiments, the cell density is maintained ata concentration of between about 1.0×10⁶ and about 4.0×10⁶ cells/ml foran extended period of time. In other embodiments, the cell density ismaintained at a concentration of between about 1.0×10⁶ and about 4.0×10⁶cells/ml for an extended period of time. In yet other embodiments, thecell density may be maintained at a concentration between about 2.0×10⁶and about 4.0×10⁶, or between about 1.0×10⁶ and about 2.5×10⁶, orbetween about 1.5×10⁶ and about 3.5×10⁶, or any other similar range, foran extended period of time. The cell density at which a cell-culture ismaintained at for production of a recombinant ADAMTS protein (e.g.,ADAMTS13) will depend upon the culture-conditions and medium used forprotein expression. One of skill in the art will readily be able todetermine the optimal cell density for a cell-culture producing anADAMTS protein. In certain embodiments, the culture is maintained for atleast 7 days, or at least 14 days, 21 days, 28 days, or at least 5weeks, 6 weeks, 7 weeks, or at least 2 months, or 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18 months or longer. In someembodiments, the cultures may be performed as suspension batch cultures.In other embodiments, the cultures may be performed as adherent batchcultures. In certain embodiments, the medium may be an animalprotein-free, oligopeptide-free, or chemically defined medium. In apreferred embodiment, the ADAMTS protein is ADAMTS13.

In other embodiments, the methods of ADAMTS13 expression allow for theproduction of at least at or about 500 units of ADAMTS13 activity perliter culture per day (500 U/L/D), for example FRETS-VWF73 activityunits, with a specific activity of at least at or about 500 U/mg A13. Inanother embodiments, the methods of ADAMTS13 expression allow for theproduction of at least at or about 600 U/L/D. In another embodiments,the methods of ADAMTS13 expression allow for the production of at leastat or about 700 U/L/D. In another embodiments, the methods of ADAMTS13expression allow for the production of at least at or about 800 U/L/D.In another embodiments, the methods of ADAMTS13 expression allow for theproduction of at least at or about 900 U/L/D. In another embodiments,the methods of ADAMTS13 expression allow for the production of at leastat or about 1000 U/L/D. In another embodiments, the methods of ADAMTS13expression allow for the production of at least at or about 1100 U/L/D.In another embodiments, the methods of ADAMTS13 expression allow for theproduction of at least at or about 1200 U/L/D. In another embodiments,the methods of ADAMTS13 expression allow for the production of at leastat or about 1300 U/L/D. In another embodiments, the methods of ADAMTS13expression allow for the production of at least at or about 1400 U/L/D.In another embodiments, the methods of ADAMTS13 expression allow for theproduction of at least at or about 1500 U/L/D. In yet anotherembodiments, the methods of ADAMTS13 expression allow for the productionof at least at or about 2000 U/L/D. In some embodiments, the culturesmay be performed as suspension batch cultures. In other embodiments, thecultures may be performed as adherent batch cultures. In certainembodiments, the medium may be an animal protein-free,oligopeptide-free, or chemically defined medium. In a preferredembodiment, the ADAMTS protein is ADAMTS13.

In another embodiment, the methods allow for the extended expression ofADAMTS13 protein with high specific activities, for example, a specificactivity of at least about 600U/mg A13 protein, or at least about 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, or more U/mg A13 protein for an extended period of time. Incertain embodiments, the culture is maintained for at least 7 days, orat least 14 days, 21 days, 28 days, or at least 5 weeks, 6 weeks, 7weeks, or at least 2 months, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18 months or longer. In some embodiments, the cultures maybe performed as suspension batch cultures. In other embodiments, thecultures may be performed as adherent batch cultures. In certainembodiments, the medium may be an animal protein-free,oligopeptide-free, or chemically defined medium. In a preferredembodiment, the ADAMTS protein is ADAMTS13.

3. Culture Conditions

Surprisingly, it has also been found that cell viabilities and ADAMTS13activities can be increased by small variations in the temperature andpH of the cell culture. As can be seen in FIG. 2, the specific activityof the expressed ADAMTS13 is greatly enhanced when cells harboring anucleotide encoding an ADAMTS13 protein are cultured at a pH below about7.30. Temperature, although to a lesser degree, is also a factorcontributing to ADAMTS13 specific activity in these studies. Similarly,it was found that the volumetric productivity of A13 (measured byFRETS-VWF73) in the supernatants of cultures grown at a pH of betweenabout 6.80 and about 7.3 was greatly increased as compared to pH levelsabove and below that range. ADAMTS13 productivity was also affected bythe temperature of the culture. Maximum activity was found in culturesgrown at temperature between about 34° C. and about 37° C.

In other embodiments, the methods of ADAMTS protein expression comprisea step of maintaining the temperature of the culture medium at atemperature of about 34° C. and about 37° C. In certain embodiments, theculture medium may be maintained at a temperature of 36.5° C. or less,36.0° or less, 35.5° C. or less, or less than 35.0° C. In a specificembodiment, the temperature is maintained at a temperature of about 36°C. In a specific embodiment the culture is maintained at a combinationof the temperature and pH ranges mentioned above, e.g. 36.5° C. or lessand a pH of e.g. 7.15 or less. In a preferred embodiment the culture ismaintained at a temperature of 36.0° C. and a pH of 7.10.

Accordingly, in some embodiments, the methods of ADAMTS proteinexpression comprise a step of maintaining the pH of the culture mediumat a pH of between about 6.8 and 7.3. In certain embodiments, the pH maybe between about 7.0 and about 7.25 or between about 7.05 and about7.15. In certain embodiments the ph may be maintained at a pH of 7.20 orless, 7.15 or less, 7.10 or less or 7.05 or less. In one specificembodiment, the pH of the culture medium is maintained at a pH of about7.1.

In one embodiment, the invention provides a method for the expressing anADAMTS protein comprises culturing a cell harboring a nucleic acidencoding an ADAMTS protein (e.g., ADAMTS13) at a temperature at or aboutbetween 34° C. and about 37° C. at a pH of at or about between 6.8 and7.3. In another embodiment, the temperature of the culture will be at orabout between 35° C. and about 37° C. and the pH will be at or aboutbetween 6.8 and 7.3. In another embodiment, the temperature of theculture will be at or about between 35.5° C. and about 36.5° C. and thepH will be at or about between 6.8 and 7.3. In another embodiment, thetemperature of the culture will be at or about 36° C. and the pH will beat or about between 6.8 and 7.3. In one embodiment, the culture mediumwill be supplemented with zinc. In another specific embodiment, theculture medium will be supplemented with calcium. In another specificembodiment, the culture medium will be supplemented with nicotinamide(vitamin B3). In one embodiment, the culture medium will be supplementedwith zinc and calcium. In one embodiment, the zinc and calciumconcentrations will be one of Var. 94 to Var. 113 (Table 7). In anotherembodiment, the culture medium will be supplemented with zinc andnicotinamide (vitamin B3). In one embodiment the zinc and nicotinamide(vitamin B3) concentrations will be one of Var. 114 to Var. 133 (Table8). In another embodiment, the culture medium will be supplemented withcalcium and nicotinamide (vitamin B3). In one embodiment, the calciumand nicotinamide (vitamin B3) concentrations will be one of Var. 134 toVar. 149 (Table 9). In yet another embodiment, the culture medium willbe concentrated with zinc, calcium, and nicotinamide (vitamin B3). Inone embodiment, the zinc, calcium, and nicotinamide (vitamin B3)concentrations will be one of Var. 14 to Var. 93 (Table 3 to Table 6).

In another embodiment, the invention provides a method for theexpressing an ADAMTS protein comprises culturing a cell harboring anucleic acid encoding an ADAMTS protein (e.g., ADAMTS13) at atemperature at or about between 34° C. and about 37° C. at a pH of at orabout between 6.9 and 7.25. In another embodiment, the temperature ofthe culture will be at or about between 35° C. and about 37° C. and thepH will be at or about between 6.9 and 7.25. In another embodiment, thetemperature of the culture will be at or about between 35.5° C. andabout 36.5° C. and the pH will be at or about between 6.9 and 7.25. Inanother embodiment, the temperature of the culture will be at or about36° C. and the pH will be at or about between 6.9 and 7.25. In oneembodiment, the culture medium will be supplemented with zinc. Inanother specific embodiment, the culture medium will be supplementedwith calcium. In another specific embodiment, the culture medium will besupplemented with nicotinamide (vitamin B3). In one embodiment, theculture medium will be supplemented with zinc and calcium. In oneembodiment, the zinc and calcium concentrations will be one of Var. 94to Var. 113 (Table 7). In another embodiment, the culture medium will besupplemented with zinc and nicotinamide (vitamin B3). In one embodimentthe zinc and nicotinamide (vitamin B3) concentrations will be one ofVar. 114 to Var. 133 (Table 8). In another embodiment, the culturemedium will be supplemented with calcium and nicotinamide (vitamin B3).In one embodiment, the calcium and nicotinamide (vitamin B3)concentrations will be one of Var. 134 to Var. 149 (Table 9). In yetanother embodiment, the culture medium will be concentrated with zinc,calcium, and nicotinamide (vitamin B3). In one embodiment, the zinc,calcium, and nicotinamide (vitamin B3) concentrations will be one ofVar. 14 to Var. 93 (Table 3 to Table 6).

In another embodiment, the invention provides a method for theexpressing an ADAMTS protein comprises culturing a cell harboring anucleic acid encoding an ADAMTS protein (e.g., ADAMTS13) at atemperature at or about between 34° C. and about 37° C. at a pH of at orabout between 7.0 and 7.20. In another embodiment, the temperature ofthe culture will be at or about between 35° C. and about 37° C. and thepH will be at or about between 7.0 and 7.20. In another embodiment, thetemperature of the culture will be at or about between 35.5° C. andabout 36.5° C. and the pH will be at or about between 7.0 and 7.20. Inanother embodiment, the temperature of the culture will be at or about36° C. and the pH will be at or about between 7.0 and 7.20. In oneembodiment, the culture medium will be supplemented with zinc. Inanother specific embodiment, the culture medium will be supplementedwith calcium. In another specific embodiment, the culture medium will besupplemented with nicotinamide (vitamin B3). In one embodiment, theculture medium will be supplemented with zinc and calcium. In oneembodiment, the zinc and calcium concentrations will be one of Var. 94to Var. 113 (Table 7). In another embodiment, the culture medium will besupplemented with zinc and nicotinamide (vitamin B3). In one embodimentthe zinc and nicotinamide (vitamin B3) concentrations will be one ofVar. 114 to Var. 133 (Table 8). In another embodiment, the culturemedium will be supplemented with calcium and nicotinamide (vitamin B3).In one embodiment, the calcium and nicotinamide (vitamin B3)concentrations will be one of Var. 134 to Var. 149 (Table 9). In yetanother embodiment, the culture medium will be concentrated with zinc,calcium, and nicotinamide (vitamin B3). In one embodiment, the zinc,calcium, and nicotinamide (vitamin B3) concentrations will be one ofVar. 14 to Var. 93 (Table 3 to Table 6).

The concept of cell-growth on microcarriers was first described by vanWezel (van Wezel AL., Nature 1967 Oct 7;216(5110):64-5) and allows forcell attachment on the surface of small solid particles suspended in thegrowth medium. These methods provide for high surface-to-volume ratiosand thus allow for efficient nutrient utilization. Furthermore, forexpression of secreted proteins in eukaryotic cell lines, the increasedsurface-to-volume ratio allows for higher levels of secretion and thushigher protein yields in the supernatant of the culture. Finally, thesemethods allow for the easy scale-up of eukaryotic expression cultures.

The cells expressing an ADAMTS protein can be bound to a spherical or aporous microcarrier during cell culture growth. The microcarrier can bea microcarrier selected from the group of microcarriers based ondextran, collagen, plastic, gelatine and cellulose and others asdescribed in Butler (1988. In: Spier & Griffiths, Animal cellBiotechnology 3:283-303). Therefore, according to one embodiment of theinvention cells expressing an ADAMTS protein are cultured on sphericalmicrocarriers. According to another embodiment of the invention cellsexpressing an ADAMTS protein are cultured on porous microcarriers. It isalso possible to grow the cells to a biomass on a sphericalmicrocarriers and subculture the cells when they have reached finalfermenter biomass and prior to production of the expressed protein on aporous microcarrier or vice versa. Spherical microcarrier are thoseselected from the group of smooth surface such as Cytodex™ 1, Cytodex™2, and Cytodex™ 3 (GE Healthcare) and macroporous microcarriers such asCytopore™ 1, Cytopore™ 2, Cytoline™ 1, and Cytoline™ 2 (GE Healthcare).

IV. CULTURE MEDIUMS

In one aspect, the present invention provides culture mediums that areuseful for the expression of ADAMTS proteins having high specificactivities. Advantageously, it has been found that by supplementing aculture medium with various components, such as combinations of zinc,calcium, and nicotinamide (vitamin B3), that the activities of ADAMTS(e.g., ADAMTS13)enzymes expressed in cells cultured in the medium aregreatly enhanced, while the enzymes are expressed at levels as high, ifnot higher, that cells cultured in non-supplemented mediums.

Methods of preparing animal protein-free and chemically defined culturemediums are known in the art, for example in U.S. Pat. Nos. 6,171,825and 6,936,441, WO 2007/077217, and U.S. Patent Application PublicationNumbers 2008/0009040 and 2007/0212770, the disclosures of which areincorporated herein by reference in their entireties for all purposes.In one embodiment, the culture medium used in the methods describedherein is animal protein-free or oligopeptide-free medium. In certainembodiments, the culture medium may be chemically defined. In certainembodiments, the culture media may contain at least one polyamine at aconcentration of about 0.5 mg/L to about 10 mg/L.

Accordingly, in one embodiment, culture medium is provided that issupplemented with additional calcium, zinc, and/or vitamin B3. Incertain embodiments, the medium may be an animal protein-free,oligopeptide-free, or chemically defined medium. In certain embodiments,the animal protein-free or oligopeptide free medium is prepared astaught in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007/077217, andU.S. Patent Application Publication Numbers 2008/0009040 and2007/0212770, the disclosures of which are incorporated herein byreference in their entireties for all purposes, both of which areincorporated herein by reference in their entireties for all purposes,and supplemented with additional calcium, zinc, and/or vitamin B3. In aspecific embodiment, the chemically defined culture medium may besimilar to a Dulbecco's Modified Eagle's Media (DMEM), which has beensupplemented with additional calcium, zinc, and/or vitamin B3, in orderto increase the specific activity of an ADAMTS protein expressed in acell cultured in the medium. In yet other embodiments, the culturemedium is animal component free. In another embodiment, the culturemedium contains protein, e.g., animal protein from serum such as fetalcalf serum. In another embodiment, the culture has recombinant proteinsexogenously added. In another embodiment, the proteins are from acertified pathogen free animal.

In certain embodiments, the culture media contains at least onepolyamine at a concentration of at or about between 0.5 mg/L and 30mg/L. In another embodiment, the culture medium contains at least onepolyamine at or about between 0.5 mg/L and 10 mg/L. In one embodiment,the culture medium contains at least one polyamine at or about between 2mg/L and 8 mg/L. In certain embodiments the polyamine is from the groupof ornithine, putrescine, spermine or spermidine, or the like. In apreferred embodiment, the polyamine is putrescine. In a specificembodiment, the culture medium contains at or about between 2 mg/L and 8mg/L putrescine.

In one embodiment, a culture medium is provided for the expression of anADAMTS protein (e.g., ADAMTS13) containing at least at or about 2 μMzinc. In another embodiment, the culture medium contains at least at orabout 5 μM zinc. In one embodiment, the culture medium contains at orabout between 2 μM and 12 μM zinc. In another embodiment, the culturemedium contains at or about between 5 μM and 12 μM zinc. In yet otherembodiments, the culture medium may contain at least at or about 2 μM,or at least at or about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM,11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, or more zinc.

Generally, any zinc salt may be used to supplement the mediums of theinvention, non-limiting examples of acceptable salts include,ZnSO₄.7H₂O, ZnSO₃.2H₂O, (C₆H₅O₇)₂Zn₃.2H₂O, ZnBr₂, ZnBr₂.2H₂O, ZnCl₂, Zn(NO₃)₂.6H₂O, Zn (H₂PO₄)2.H₂O, (C₂H₃O₂)₂Zn.2H₂O, and the like. In certainembodiments, a pharmaceutically acceptable salt of zinc is used tosupplement the culture mediums of the invention. In other embodiments, azinc containing peptide or protein preparation, for example insulin, maybe used to the supplement the culture provided herein.

In another embodiment, a culture medium is provided for the expressionof an ADAMTS protein (e.g., ADAMTS13) containing at least at or about0.5 mM calcium. In another embodiment, the culture medium contains atleast at or about 1.5 mM calcium. In one embodiment, the culture mediumcontains at or about between 0.5 mM and 1.5 mM calcium. In yet otherembodiments, the culture medium may contain at least at or about 0.5 mM,or at least at or about 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM,1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM,2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, ormore calcium.

Generally, any calcium salt may be used to supplement the mediums of theinvention, non-limiting examples of acceptable salts include CaC₁₂,CaC₁₂, CaFPO₃.2H₂O, CaI₂, CaBr₂, (C₂H₃)O₂)₂Ca, (CHO₂)₂Ca, (C₆H₇O₆)₂Ca,(C₆H₅O₇)₂Ca₃.2H₂O, and the like. In certain embodiments, apharmaceutically acceptable salt of calcium is used to supplement theculture mediums of the invention.

In another embodiment, a culture medium is provided for the expressionof an ADAMTS protein (e.g., ADAMTS13) containing at least at or about 2mg/L nicotinamide (vitamin B3). In another embodiment, the culturemedium contains at least at or about 7 mg/L nicotinamide (vitamin B3).In one embodiment, the culture medium contains at or about between 2mg/L and 10 mg/L nicotinamide (vitamin B3). In yet other embodiments,the culture medium may contain at least at or about 2 mg/L, 3 mg/L, 4mg/L, 5 mg/L, 6 mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L,or higher concentrations of nicotinamide (vitamin B3).

Advantageously, it has been found that by supplementing a culture mediumwith both zinc and calcium, the specific activity of ADAMTS13 proteinexpressed in the culture medium is greatly increased. Accordingly, inone embodiment, the culture mediums provided herein for the expressionof an ADAMTS protein (e.g., ADAMTS13) may be supplemented with both zincand calcium. For example, a culture medium may be supplemented with zincand calcium at levels provided in Table 7, i.e., at a level according toany one of Var. 94 to Var. 113.

TABLE 7 Exemplary embodiments of a culture mediums supplemented withboth zinc and calcium, which are useful for the expression of anADAMTS13 protein. At least 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0mM, Between 2.25 mM, 2.5 mM, 2.75 mM, At least At least 0.5 mM and 3.0mM, 3.5 mM, 4.0 mM, 0.5 mM 1.5 mM 1.5 mM 4.5 mM, 5.0 mM, or more calciumcalcium calcium calcium At least 2 μM Zinc Var. 94  Var. 95  Var. 96 Var. 97  At least 5 μM Zinc Var. 98  Var. 99  Var. 100 Var. 101 Between2 μM and 12 μM Var. 102 Var. 103 Var. 104 Var. 105 zinc Between 5 μM and12 μM Var. 106 Var. 107 Var. 108 Var. 109 zinc At least 3 μM, 4 μM, 5μM, Var. 110 Var. 111 Var. 112 Var. 113 6 μM, 7 μM, 8 μM, 9 μM, 10 μM,11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, or more zinc*Var. = Variation

Similarly, it has been found that by supplementing a culture medium withboth zinc and nicotinamide (vitamin B3), the specific activity ofADAMTS13 protein expressed in the culture medium is synergisticallyincreased. For example, this effect can be seen in Example 2 (compareTable 14 day 11 with Table 13 days 4 and 7). Accordingly, in oneembodiment, the culture mediums provided herein for the expression of anADAMTS protein (e.g., ADAMTS13) may be supplemented with both zinc andnicotinamide (vitamin B3). For example, a culture medium may besupplemented with zinc and nicotinamide (vitamin B3) at levels providedin Table 8, i.e., at a level according to any one of Var. 114 to Var.133.

TABLE 8 Exemplary embodiments of a culture mediums supplemented withboth zinc and nicotinamide (vitamin B3), which are useful for theexpression of an ADAMTS13 protein. At least 3 mg/L, 4 mg/L, 5 mg/L, 6mg/L, 7 mg/L, Between 8 mg/L, 9 mg/L, 10 mg/L, At least At least 2 mg/Land 15 mg/L, 20 mg/L, 2 mg/L 7 mg/L 7 mg/L or more nicotinamidenicotinamide nicotinamide nicotinamide At least 2 μM Zinc Var. 114 Var.115 Var. 116 Var. 117 At least 5 μM Zinc Var. 118 Var. 119 Var. 120 Var.121 Between 2 μM and 12 μM Var. 122 Var. 123 Var. 124 Var. 125 zincBetween 5 μM and 12 μM Var. 126 Var. 127 Var. 128 Var. 129 zinc At least3 μM, 4 μM, 5 μM, Var. 130 Var. 131 Var. 132 Var. 133 6 μM, 7 μM, 8 μM,9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, ormore zinc Var. = Variation

Advantageously, it has been found that by supplementing a culture mediumwith both nicotinamide and calcium, the specific activity of ADAMTS13protein expressed in the culture medium is greatly increased.Accordingly, in one embodiment, the culture mediums provided herein forthe expression of an ADAMTS protein (e.g., ADAMTS13) may be supplementedwith both nicotinamide (vitamin B3) and calcium. For example, a culturemedium may be supplemented with nicotinamide (vitamin B3) and calcium atlevels provided in Table 9, i.e., at a level according to any one ofVar. 134 to Var. 149.

TABLE 9 Exemplary embodiments of a culture mediums supplemented withboth nicotinamide (vitamin B3) and calcium, which are useful for theexpression of an ADAMTS13 protein. At least 3 mg/L, 4 mg/L, 5 mg/L, 6mg/L, 7 mg/L, 8 mg/L, 9 mg/L, Between 10 mg/L, 15 mg/L, At least Atleast 2 mg/L and 20 mg/L, 2 mg/L 7 mg/L 7 mg/L or more nicotinamidenicotinamide nicotinamide nicotinamide At least 0.5 mM calcium Var. 134Var. 135 Var. 136 Var. 137 At least 1.5 mM calcium Var. 138 Var. 139Var. 140 Var. 141 Between 0.5 mM and 1.5 mM Var. 142 Var. 143 Var. 144Var. 145 calcium At least 0.6 mM, 0.7 mM, Var. 146 Var. 147 Var. 148Var. 149 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM,1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, or more calcium Var. = Variation

In some embodiments, the culture medium provided by the invention may beprovided in a liquid or a dry or powder form. The medium may bepre-aliquoted in an amount suitable for single use or provided in alarger quantity that may be used for more than one cell-culture.Generally, the medium of the invention will be provided in a sterilefashion. Accordingly, the present invention also provides kits for theexpression or production of an ADAMTS13 protein, the kits comprising aculture medium suitable for the expression of an ADAMTS protein havinghigh specific activity.

In one aspect, the present invention provides culture mediums useful forthe expression of ADAMTS protein (e.g., ADAMTS13) with high specificactivities. In one embodiment, the culture medium contains at leastabout 2 μM zinc. In another embodiment, the culture medium containsbetween about 2 μM to about 12 μM zinc. In yet another embodiment, theculture medium contains at least about 5 μM zinc. In one embodiment, theculture medium contains between about 5 μM to about 12 μM zinc. Inanother embodiment, the culture medium contains at least about 0.5 mMcalcium. In yet another embodiment, the culture medium contains betweenabout 0.5 mM and about 1.5 mM calcium. In one embodiment, the culturemedium contains at least about 2 μM zinc and at least about 0.5 mMcalcium.

In yet other embodiments, it has been found that the addition ofnicotinamide (vitamin B3) further enhances the expression and specificactivity of ADAMTS proteins in cell culture. In one embodiment, theculture medium further comprises at least about 2 mg/L nicotinamide(vitamin B3). In another embodiment, the culture medium furthercomprises at least about 7 mg/L nicotinamide (vitamin B3). In yetanother embodiment, the culture medium contains between about 2 mg/L andabout 10 mg/L nicotinamide (vitamin B3).

In certain embodiments, the culture medium is an animal protein freeculture medium. In another embodiment, the culture medium is achemically defined medium. In certain embodiments, the culture mediummay comprise one or more polyamines. In a particular embodiment, thepolyamine is putrescine, for example, at a concentration of at least 0.5mg/L. In a specific embodiment, the culture medium contains betweenabout 2 mg/L and about 8 mg/L putrescine.

1. Protein Free Culture Mediums

In certain aspects, the present invention provides culture mediums forthe expression of an ADAMTS protein (e.g., ADAMTS13), which are free ofexogenously added protein. “Protein free culture medium” and relatedterms refers to culture medium lacking protein that is from a sourceexogenous to or other than the cells in the culture, which naturallyshed proteins during growth. In one embodiment, a culture medium isprovided for the expression of an ADAMTS protein (e.g., ADAMTS13), whichis free of exogenously added protein (i.e., protein-free) and issupplemented with zinc, calcium, and/or nicotinamide (vitamin B3). Incertain embodiments, the protein free culture medium contains apolyamine. For example, at a concentration of at least 2 mg/L, or at orabout between 2 mg/L and 30 mg/L, or at or about between 2 mg/L and 8mg/L. In a specific embodiment, the polyamine is putrescine. Exemplaryprotein free culture mediums are taught in U.S. Pat. Nos. 6,171,825 and6,936,441, WO 2007/077217, and U.S. Patent Application PublicationNumbers 2008/0009040 and 2007/0212770, the disclosures of which areincorporated herein by reference in their entireties for all purposes.

In one embodiment, a protein-free culture medium is provided for theexpression of an ADAMTS protein (e.g., ADAMTS13) contains at least at orabout 2 μM zinc, at least at or about 5 μM zinc, at or about between 2μM and 12 μM zinc, or at or about between 5 μM and 12 μM zinc. In yetother embodiments, a protein-free culture medium is provided for theexpression of an ADAMTS protein (e.g., ADAMTS13) containing at least ator about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM,13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, or more zinc.

In another embodiment, a protein-free culture medium is provided for theexpression of an ADAMTS protein (e.g., ADAMTS13) contains at least at orabout 0.5 mM calcium, at least at or about 1.5 mM calcium, or at orabout between 0.5 and 1.5 mM calcium. In yet other embodiments, aprotein-free culture medium is provided for the expression of an ADAMTSprotein (e.g., ADAMTS13) containing at least at or about 0.6 mM, 0.7 mM,0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM,1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5mM, 4.0 mM, 4.5 mM, 5.0 mM, or more calcium.

In yet another embodiment, a protein-free culture medium is provided forthe expression of an ADAMTS protein (e.g., ADAMTS13) contains at leastat or about 2 mg/L nicotinamide (vitamin B3), at least at or about 7mg/L nicotinamide (vitamin B3), or at or about between 2 mg/Lnicotinamide (vitamin B3). In yet other embodiments, a protein-freeculture medium is provided for the expression of an ADAMTS protein(e.g., ADAMTS13) containing at least at or about 3 mg/L, 4 mg/L, 5 mg/L,6 mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or morenicotinamide (vitamin B3).

2. Oligopeptide Free Culture Mediums

In certain aspects, the present invention provides culture mediums forthe expression of an ADAMTS protein (e.g., ADAMTS13), which are free ofexogenously added oligopeptides. In one embodiment, a culture medium isprovided for the expression of an ADAMTS protein (e.g., ADAMTS13), whichis free of exogenously added oligopeptides (i.e., polypeptide-free) andis supplemented with zinc, calcium, and/or nicotinamide (vitamin B3). Incertain embodiments, the oligopeptide free culture medium contains apolyamine. For example, at a concentration of at least 2 mg/L, or at orabout between 2 mg/L and 30 mg/L, or at or about between 2 mg/L and 8mg/L. In a specific embodiment, the polyamine is putrescine. Exemplaryoligopeptide free culture mediums are taught in U.S. Pat. Nos. 6,171,825and 6,936,441, WO 2007/077217, and U.S. Patent Application PublicationNumbers 2008/0009040 and 2007/0212770, the disclosures of which areincorporated herein by reference in their entireties for all purposes.

In one embodiment, an oligopeptide free culture medium is provided forthe expression of an ADAMTS protein (e.g., ADAMTS13) contains at leastat or about 2 μM zinc, at least at or about 5 μM zinc, at or aboutbetween 2 μM and 12 μM zinc, or at or about between 5 μM and 12 μM zinc.In yet other embodiments, an oligopeptide free culture medium isprovided for the expression of an ADAMTS protein (e.g., ADAMTS13)containing at least at or about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, ormore zinc.

In another embodiment, an oligopeptide free culture medium is providedfor the expression of an ADAMTS protein (e.g., ADAMTS13) contains atleast at or about 0.5 mM calcium, at least at or about 1.5 mM calcium,or at or about between 0.5 and 1.5 mM calcium. In yet other embodiments,an oligopeptide free culture medium is provided for the expression of anADAMTS protein (e.g., ADAMTS13) containing at least at or about 0.6 mM,0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM,1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, or more calcium.

In yet another embodiment, an oligopeptide free culture medium isprovided for the expression of an ADAMTS protein (e.g., ADAMTS13)contains at least at or about 2 mg/L nicotinamide (vitamin B3), at leastat or about 7 mg/L nicotinamide (vitamin B3), or at or about between 2mg/L nicotinamide (vitamin B3). In yet other embodiments, anoligopeptide free culture medium is provided for the expression of anADAMTS protein (e.g., ADAMTS13) containing at least at or about 3 mg/L,4 mg/L, 5 mg/L, 6 mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20mg/L, or more nicotinamide (vitamin B3).

3. Serum Free Culture Mediums

In certain aspects, the present invention provides culture mediums forthe expression of an ADAMTS protein (e.g., ADAMTS13), which are free ofserum. In one embodiment, a culture medium is provided for theexpression of an ADAMTS protein (e.g., ADAMTS13), which is free ofexogenously added serum (i.e., serum-free) and is supplemented withzinc, calcium, and/or nicotinamide (vitamin B3). In certain embodiments,the serum-free culture medium contains a polyamine. For example, at aconcentration of at least 2 mg/L, or at or about between 2 mg/L and 30mg/L, or at or about between 2 mg/L and 8 mg/L. In a specificembodiment, the polyamine is putrescine. Exemplary serum-free culturemediums are taught in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO2007/077217, and U.S. Patent Application Publication Numbers2008/0009040 and 2007/0212770, the disclosures of which are incorporatedherein by reference in their entireties for all purposes.

In one embodiment, a serum-free culture medium is provided for theexpression of an ADAMTS protein (e.g., ADAMTS13) contains at least at orabout 2 μM zinc, at least at or about 5 μM zinc, at or about between 2μM and 12 μM zinc, or at or about between 5 μM and 12 μM zinc. In yetother embodiments, a serum-free culture medium is provided for theexpression of an ADAMTS protein (e.g., ADAMTS13) containing at least ator about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM,13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, or more zinc.

In another embodiment, a serum-free culture medium is provided for theexpression of an ADAMTS protein (e.g., ADAMTS13) contains at least at orabout 0.5 mM calcium, at least at or about 1.5 mM calcium, or at orabout between 0.5 and 1.5 mM calcium. In yet other embodiments, aserum-free culture medium is provided for the expression of an ADAMTSprotein (e.g., ADAMTS13) containing at least at or about 0.6 mM, 0.7 mM,0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM,1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5mM, 4.0 mM, 4.5 mM, 5.0 mM, or more calcium.

In yet another embodiment, a serum-free culture medium is provided forthe expression of an ADAMTS protein (e.g., ADAMTS13) contains at leastat or about 2 mg/L nicotinamide (vitamin B3), at least at or about 7mg/L nicotinamide (vitamin B3), or at or about between 2 mg/Lnicotinamide (vitamin B3). In yet other embodiments, a serum-freeculture medium is provided for the expression of an ADAMTS protein(e.g., ADAMTS13) containing at least at or about 3 mg/L, 4 mg/L, 5 mg/L,6 mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or morenicotinamide (vitamin B3).

4. Animal Protein Free Culture Mediums

In certain aspects, the present invention provides culture mediums forthe expression of an ADAMTS protein (e.g., ADAMTS13), which are free ofanimal proteins. In one embodiment, a culture medium is provided for theexpression of an ADAMTS protein (e.g., ADAMTS13), which is free ofexogenously added animal proteins or polypeptides (i.e., animal proteinfree) and is supplemented with zinc, calcium, and/or nicotinamide(vitamin B3). In certain embodiments, the animal protein free culturemedium contains a polyamine. For example, at a concentration of at least2 mg/L, or at or about between 2 mg/L and 30 mg/L, or at or aboutbetween 2 mg/L and 8 mg/L. In a specific embodiment, the polyamine isputrescine. Exemplary animal protein free culture mediums are taught inU.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007/077217, and U.S. PatentApplication Publication Numbers 2008/0009040 and 2007/0212770, thedisclosures of which are incorporated herein by reference in theirentireties for all purposes.

In one embodiment, an animal protein free culture medium is provided forthe expression of an ADAMTS protein (e.g., ADAMTS13) contains at leastat or about 2 μM zinc, at least at or about 5 μM zinc, at or aboutbetween 2 μM and 12 μM zinc, or at or about between 5 μM and 12 μM zinc.In yet other embodiments, an animal protein free culture medium isprovided for the expression of an ADAMTS protein (e.g., ADAMTS13)containing at least at or about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, ormore zinc.

In another embodiment, an animal protein free culture medium is providedfor the expression of an ADAMTS protein (e.g., ADAMTS13) contains atleast at or about 0.5 mM calcium, at least at or about 1.5 mM calcium,or at or about between 0.5 and 1.5 mM calcium. In yet other embodiments,an animal protein free culture medium is provided for the expression ofan ADAMTS protein (e.g., ADAMTS13) containing at least at or about 0.6mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM,3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, or more calcium.

In yet another embodiment, an animal protein free culture medium isprovided for the expression of an ADAMTS protein (e.g., ADAMTS13)contains at least at or about 2 mg/L nicotinamide (vitamin B3), at leastat or about 7 mg/L nicotinamide (vitamin B3), or at or about between 2mg/L nicotinamide (vitamin B3). In yet other embodiments, an animalprotein free culture medium is provided for the expression of an ADAMTSprotein (e.g., ADAMTS13) containing at least at or about 3 mg/L, 4 mg/L,5 mg/L, 6 mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, ormore nicotinamide (vitamin B3).

V. ADAMTS PROTEIN PURIFICATION

ADAMTS13 is secreted into the plasma in vivo, where it functions toregulate clotting activity by cleaving large multimers of vWF. Theability of mammalian cells to secrete ADAMTS13 after expression can beexploited during the production and purification of ADAMTS13compositions in cell culture. For example, by expressing recombinantADAMTS13 in mammalian cell culture, ADAMTS13 compositions can readily berecovered directly from the culture supernatant without the need toharvest and lyse cells. This permits the use of techniques such ascontinuous cell culture (e.g., perfusion or chemostatic cell culture) toproduce large amounts of the protein without multiple culture lag andrecovery periods. In one aspect of the invention, methods are providedfor the purification of ADAMTS proteins having high specific activityfrom cell culture.

Accordingly, in one embodiment, ADAMTS13 proteins are expressed inculture and recovered directly from the culture supernatant. In thisfashion, ADAMTS13 proteins are recovered by removing a fraction of theculture and purifying ADAMTS13 away from the other components of thesupernatant. Generally, this involves separating any cells that arerecovered along with the supernatant by filtration or centrifugation andsubjecting the supernatant to one or more ADAMTS13 purification steps.

U.S. Patent Application Publication Number 2005/0266528 and Zheng etal., (2001, Blood, 98:1662-1666), the disclosures of which are hereinincorporated by reference in their entireties for all purposes) provideexemplary methods for purifying ADAMTS13. Purified ADAMTS13 may beformulated according to conventional methods and used therapeutically,for example, to treat TTP.

In one aspect, the present invention provides methods for producing anADAMTS protein composition (e.g., an ADAMTS13 composition). In a firstembodiment, the method comprises the steps of: (a) culturing a cellharboring a nucleic acid encoding an ADAMTS protein in an animalprotein-free culture medium; (b) removing a fraction of the supernatantfrom the culture; (c) performing a filtration or centrifugation step toremove any residual cells; (d) performing an ultrafiltration step toconcentrate the ADAMTS protein; and (e) performing a diafiltration stepwith a buffer comprising at least about 0.5 μM zinc and at least about0.1 mM calcium; thereby preparing an ADAMTS composition.

In certain embodiments, the step of culturing a cell comprises batchcell cultivation. In other embodiments, the step of culturing a cellcomprises continuous cell cultivation.

In certain embodiments, the culture medium contains calcium. In aspecific embodiment, the culture medium contains at least 0.5 mMcalcium. In other embodiments, the culture medium contains zinc. In aspecific embodiment, the culture medium contains at least 2 μM zinc. Inyet other embodiments, the culture medium contains nicotinamide (vitaminB3). In a specific embodiment, the culture medium contains at least 2mg/L nicotinamide (vitamin B3). In certain embodiments, thediafiltration buffer contains at least about 5 μM zinc and at leastabout 2 mM calcium.

In certain embodiments, less than about 20% of the ADAMTS13 specificactivity is lost between the end of step (c) and the end of step (e). Inother embodiments, less than about 10% of the ADAMTS13 specific activityis lost between the end of step (c) and the end of step (e). In yetother embodiments, the ADAMTS13 composition has a specific activity ofat least about 1000 U/mg. In another embodiment, the ADAMTS13composition has a specific activity of at least about 1500 U/mg.

A. Buffer Exchange

Typically, when purifying a secreted protein from a culture supernatant,the first step of the purification process involves exchanging theculture medium for a buffered solution, which facilitates furtherpurification of the protein of interest. Several options exist forexchanging the culture medium for a buffer, including withoutlimitation, diafiltration, dialysis, buffer exchange techniques, gelfiltration, chromatography, and the like.

It was found that ADAMTS13 protein compositions lost a significantfraction of their specific activity during such purification steps thatrequire the introduction of new buffers, e.g., diafiltration, dialysis,buffer exchange, chromatography, and similar steps, regardless of thelength of time between harvesting the supernatant from the culture andthe purification step. Advantageously, however, the present inventorshave discovered that by including zinc and calcium in the buffer beingintroduced into the system, such as in diafiltration, dialysis, bufferexchange, gel filtration, and chromatographic steps, that the highspecific activities of the ADAMTS13 composition is retained. As evidenceof this, Example 6 demonstrates that diafiltration of an ADAMTS13composition with a buffer lacking calcium and zinc results in an averageloss of almost 25% of the specific activity of the composition, whileinclusion of calcium and zinc almost entirely prevent this loss (Table22). Accordingly, the present invention provides methods for reducingthe loss of activity for an ADAMTS protein compositions afterdiafiltration, or similar methods, e.g., dialysis, buffer exchange,chromatography, by the inclusion of zinc and calcium into the buffersystem.

In one embodiment, a method is provided for purifying an ADAMTS protein(e.g., ADAMTS13) composition, the method comprising the steps of (a)culturing a cell harboring a nucleic acid encoding an ADAMTS protein(e.g., ADAMTS13) in a culture medium; (b) recovering a portion of theculture medium supernatant containing an ADAMTS protein (e.g.,ADAMTS13); and (c) exchanging the culture medium supernatant with abuffered solution containing zinc and calcium, thereby preparing anADAMTS protein (e.g., ADAMTS13) composition. In one embodiment, theculture medium contains zinc, calcium, and optionally nicotinamide(vitamin B3). In a preferred embodiment, the step of culturing a cellcomprises a continuous culture (e.g., perfusion or chemostatic culture).In another preferred embodiment, the culture is maintained at atemperature between 34° C. and 37° C. In yet another preferredembodiment, the culture is maintained at a pH between 6.9 and 7.2.

In one specific embodiment, the step of exchanging the culture mediumsupernatant with a buffered solution comprises the use of a buffercontaining at least at or about 0.5 mM calcium and at least at or about0.5 μM zinc. In another embodiment, the buffer contains at least at orabout 2 mM calcium and at least at or about 5 μM zinc. In certainembodiments, the concentration of calcium may be at least about 0.1 mM,0.3 mM, 0.5 mM, 0,75 mM, 1 mM, 1.5 mM, 2 mM, 3 mM, 5 mM or more calcium.In certain embodiments, the concentration of zinc may be at least about0.5 μM, 1 μM, 2 μM, 3 μM, 5 μM, 10 μM or more zinc. Generally, anycombination of the concentrations above is suitable for use in thepresent methods.

In one embodiment, a method is provided for the buffer exchange of anADAMTS protein composition (e.g., ADAMTS13) by performing the bufferexchange (e.g., diafiltration, dialysis, gel filtration, etc.) with abuffer containing calcium and zinc. In one specific embodiment, themethod comprises the use of a buffer containing at least at or about 0.5mM calcium and at least at or about 0.5 μM zinc. In another embodiment,the buffer contains at least at or about 1 mM calcium and at least at orabout 1 μM zinc. In one embodiment, the buffer contains at least at orabout 2 mM calcium and at least at or about 2 μM zinc. In anotherembodiment, the buffer contains at least at or about 2 mM calcium and atleast at or about 5 μM zinc. In yet other embodiments, the buffercontains between 0.5 mM and 5 mM calcium and between 0.5 μM and 5 μMzinc. In certain embodiments, the concentration of calcium may be atleast about 0.1 mM, 0.3 mM, 0.5 mM, 0,75 mM, 1 mM, 1.5 mM, 2 mM, 3 mM, 5mM or more calcium. In certain embodiments, the concentration of zincmay be at least about 0.5 μM, 1 μM, 2 μM, 3 μM, 5 μM, 10 μM or morezinc. In a certain embodiment the harvest, cell free supernatant or thediafiltration buffer contains combinations of calcium and zinc in theabove mentioned concentrations.

In another embodiment, a method is provided for stabilizing theenzymatic activity of an ADAMTS protein (e.g., ADAMTS13) afterexpression in cell culture comprising supplementing the cell containingharvest, the cell free supernatant or a diafiltration buffer used toconcentrate or for exchange of buffer of an ADAMTS solution with calciumand zinc. In one specific embodiment, the method comprises the use of abuffer containing at least at or about 0.5 mM calcium and at least at orabout 0.5 μM zinc. In another embodiment, the buffer contains at leastat or about 2 mM calcium and at least at or about 5 μM zinc. In certainembodiments, the concentration of calcium may be at least about 0.1 mM,0.3 mM, 0.5 mM, 0,75 mM, 1 mM, 1.5 mM, 2 mM, 3 mM, 5 mM or more calcium.In certain embodiments, the concentration of zinc may be at least about0.5 μM, 1 μM, 2 μM, 3 μM, 5 μM, 10 μM or more zinc. In a certainembodiment the harvest, cell free supernatant or the diafiltrationbuffer contains combinations of calcium and zinc in the above mentionedconcentrations.

In other embodiments, the methods provided herein result in a loss ofless than 15% of the specific activity present during any individualstep. In a preferred embodiment, the methods provided herein result in aloss of less than 10% of the specific activity after present during anyindividual step. In a particular embodiment, less than 15% of thespecific activity present in the recovered culture supernatant is lostduring an initial buffer exchange step (e.g., diafiltration ordialysis). In a preferred embodiment, less than 10% of the specificactivity present in the recovered culture supernatant is lost during aninitial buffer exchange step (e.g., diafiltration or dialysis).

B. Chromatography

In certain embodiments, the ADAMTS protein (e.g., ADAMTS13) compositionis further enriched by one or more chromatographic steps. In oneembodiment, a method is provided for providing an enriched ADAMTSprotein (e.g., ADAMTS13) composition, the method comprising the steps of(a) culturing a cell harboring a nucleic acid encoding an ADAMTS protein(e.g., ADAMTS13) in a culture medium; (b) recovering a portion of theculture medium supernatant containing an ADAMTS protein (e.g.,ADAMTS13); (c) exchanging the culture medium supernatant with a bufferedsolution containing zinc and calcium, to form a first ADAMTS protein(e.g., ADAMTS13) composition; and (d) further enriching the ADAMTSprotein (e.g., ADAMTS13) with a chromatographic step, thereby providingan enriched ADAMTS (e.g., ADAMTS13) composition. In one embodiment, theculture medium contains zinc, calcium, and optionally nicotinamide(vitamin B3). In a preferred embodiment, the step of culturing a cellcomprises a continuous culture (e.g., perfusion or chemostatic culture).In another preferred embodiment, the culture is maintained at atemperature between 34° C. and 37° C. In yet another preferredembodiment, the culture is maintained at a pH between 6.9 and 7.2.

In one specific embodiment, the step of exchanging the culture mediumsupernatant with a buffered solution and/or the chromatographic stepcomprises the use of a buffer containing at least at or about 0.5 mMcalcium and at least at or about 0.5 μM zinc. In a preferred embodiment,both steps comprise the use of buffers containing at least at or about0.5 mM calcium and at least at or about 0.5 μM zinc. In anotherembodiment, the buffer contains at least at or about 2 mM calcium and atleast at or about 5 μM zinc. In certain embodiments, the concentrationof calcium may be at least about 0.1 mM, 0.3 mM, 0.5 mM, 0,75 mM, 1 mM,1.5 mM, 2 mM, 3 mM, 5 mM or more calcium. In certain embodiments, theconcentration of zinc may be at least about 0.5 μM, 1 μM, 2 μM, 3 μM, 5μM, 10 μM or more zinc. Generally, any combination of the concentrationsabove is suitable for use in the present methods.

In one embodiment, a method is provided for maintaining the specificactivity of an ADAMTS protein (e.g., ADAMTS13) composition during achromatographic step by supplementing the buffer(s) used in thechromatographic step with zinc and calcium. In one specific embodiment,the method comprises the use of a buffer containing at least at or about0.5 mM calcium and at least at or about 0.5 μM zinc. In anotherembodiment, the buffer contains at least at or about 2 mM calcium and atleast at or about 5 μM zinc. In certain embodiments, the concentrationof calcium may be at least about 0.1 mM, 0.3 mM, 0.5 mM, 0,75 mM, 1 mM,1.5 mM, 2 mM, 3 mM, 5 mM or more calcium. In certain embodiments, theconcentration of zinc may be at least about 0.5 μM, 1 μM, 2 μM, 3 μM, 5μM, 10 μM or more zinc. Generally, any combination of the concentrationsabove is suitable for use in the present methods.

Non-limiting examples of chromatographic techniques that may be used topurify an ADAMTS protein composition (e.g., an ADAMTS13 composition)include anion exchange chromatography (AEC), cation exchangechromatography (CEC), hydrophobic exchange chromatography (HIC),hydroxyapatite chromatography (HAP), immuno-affinity chromatography,size exclusion chromatography (i.e., gel filtration), or other suitablechromatographic step. Chromatographic steps may be performed in eitherbatch or column mode. In certain embodiments, buffers used to performany of these chromatographic techniques will include zinc and calcium.In one specific embodiment, the method comprises the use of a buffercontaining at least at or about 0.5 mM calcium and at least at or about0.5 μM zinc. In another embodiment, the buffer contains at least at orabout 2 mM calcium and at least at or about 5 μM zinc. In certainembodiments, the concentration of calcium may be at least about 0.1 mM,0.3 mM, 0.5 mM, 0,75 mM, 1 mM, 1.5 mM, 2 mM, 3 mM, 5 mM or more calcium.In certain embodiments, the concentration of zinc may be at least about0.5 μM, 1 μM, 2 μM, 3 μM, 5 μM, 10 μM or more zinc. Generally, anycombination of the concentrations above is suitable for use in thepresent methods.

Any suitable anion exchange resin may be used in the methods providedherein. Non-limiting examples of anion exchange resins suitable for useinclude, diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), andquaternary ammonium (Q) resins.

Any suitable cation exchange resin may be used in the methods providedherein. Non-limiting examples of cation exchange resins suitable for useinclude, carboxymethyl (CM), sulfopropyl (SP), methyl sulfonate (S)resins.

Any suitable hydroxyapatite or other calcium-based resin may be used inthe methods provided herein. Non-limiting examples of suitable resinsinclude hydroxyapatite resins, fluorapatite resins, fluorhydroxyapatiteresins, and the like.

Any suitable hydrophobic interaction chromatography resin may be used inthe methods provided herein. Non-limiting examples of suitable resinsinclude phenyl-resins, methyl-resins, butyl-resins, octyl-resins, andthe like.

In certain embodiments, an ADAMTS13 protein (e.g., ADAMTS13) may befurther enriched by immuno-affinity chromatography, for example withresins conjugated to an antibody, aptamer, or other binding moleculehighly specific for the ADAMTS13 protein (e.g.,

In one embodiment, the method of reducing the loss of ADAMTS activityresults in a net loss of less than 20% during any individual step,including but not-limited to, buffer exchange, diafiltration, dialysis,gel filtration, ion exchange chromatography, affinity chromatography,nanofiltration, ultrafiltration, sterile filtration, and the like. Inother embodiments, the methods provided herein result in a loss of lessthan 15% of the specific activity present during any individual step. Ina preferred embodiment, the methods provided herein result in a loss ofless than 10% of the specific activity after present during anyindividual step. In a particular embodiment, less than 15% of thespecific activity present in the recovered culture supernatant is lostduring an initial buffer exchange step (e.g., diafiltration ordialysis). In a preferred embodiment, less than 10% of the specificactivity present in the recovered culture supernatant is lost during aninitial buffer exchange step (e.g., diafiltration or dialysis).

C. Virus Inactivation and/or Removal

In certain embodiments, the methods provided herein for the preparationof an ADAMTS (e.g., ADAMTS13) composition will further include at leastone viral inactivation or removal steps. In certain embodiments, themethods provided herein will include at least two or at least three,viral inactivation or removal steps. Non-limiting examples of viralinactivation or removal steps that may be employed with the methodsprovided herein include, solvent detergent treatment (Horowitz et al.,Blood Coagul Fibrinolysis 1994 (5 Suppl 3): S21-S28 and Kreil et al.,Transfusion 2003 (43):1023-1028, the disclosures of which are expresslyincorporated by reference herein in their entireties for all purposes),nanofiltration (Hamamoto et al., Vox Sang 1989 (56)230-236 and Yuasa etal., J Gen Virol. 1991 (72 (pt 8)):2021-2024, the disclosures of whichare expressly incorporated by reference herein in their entireties forall purposes). In a preferred embodiment, the present invention providesmethods for the preparation of an ADAMTS (e.g., ADAMTS13) compositioncomprising solvent detergent treatment and nanofiltration.

In one embodiment, a method is provided for providing a virally safeADAMTS protein (e.g., ADAMTS13) composition, the method comprising thesteps of (a) culturing a cell harboring a nucleic acid encoding anADAMTS protein (e.g., ADAMTS13) in a culture medium; (b) recovering aportion of the culture medium supernatant containing an ADAMTS protein(e.g., ADAMTS13); (c) exchanging the culture medium supernatant with abuffered solution containing zinc and calcium, to form a first ADAMTSprotein (e.g., ADAMTS13) composition; (d) optionally further enrichingthe ADAMTS protein (e.g., ADAMTS13) with a chromatographic step; and (e)performing at least one virus inactivation or removal step, therebyproviding a virally safe ADAMTS (e.g., ADAMTS13) composition. In oneembodiment, the culture medium contains zinc, calcium, and optionallynicotinamide (vitamin B3). In a preferred embodiment, the step ofculturing a cell comprises a continuous culture (e.g., perfusion orchemostatic culture). In another preferred embodiment, the culture ismaintained at a temperature between 34° C. and 37° C. In yet anotherpreferred embodiment, the culture is maintained at a pH between 6.9 and7.2. In one embodiment, the virus removal step is nanofiltration.

1. Solvent and Detergent (S/D) Treatment

In order to inactivate various viral contaminants which may be presentin ADAMTS culture, one or more ADAMTS (e.g., ADAMTS13) intermediatesolutions may be subjected to a solvent detergent (S/D) treatment.Methods for the detergent treatment of solutions are well known in theart (for review see, Pelletier J P et al., Best Pract Res Clin Haematol.2006; 19(1):205-42, the disclosure of which is expressly incorporated byreference herein in its entirety for all purposes). Generally, anystandard S/D treatment may be used in conjunction with the methodsprovided herein. For example, an exemplary protocol for an S/D treatmentis provided below.

In one embodiment, Triton X-100, Tween-20, and tri(n-butyl)phosphate(TNBP) are added to an ADAMTS (e.g., ADAMTS13) intermediate solution atfinal concentrations of at or about 1.0%, 0.3%, and 0.3%, respectively.The mixture is then stirred at a temperature at or about between 18° C.and 25° C. for at least about an hour.

2. Nanofiltration and Ultra/Diafiltration

In order to reduce the viral load of an ADAMTS protein (e.g., ADAMTS13)composition provided herein, the composition may be nanofiltered using asuitable nanofiltration device. In certain embodiments, thenanofiltration device will have a mean pore size of at or about between15 nm and 200 nm. Examples of nanofilters suitable for this use include,without limitation, DVD, DV 50, DV 20 (Pall), Viresolve NFP®, ViresolveNFR® (Millipore), Planova® 15N, 20N, 35N, and 75N (Planova). In aspecific embodiment, the nanofilter may have a mean pore size of at orabout between 15 and 72 nm, or at or about between 19 and 35 nm, or ofat or about 15 nm, 19 nm, 20 nm, 35 nm, or 72 nm. In a preferredembodiment, the nanofilter will have a mean pore size of at or about 19nm, 20 nm, or 35 nm, such as an Asahi PLANOVA® 20N or PLANOVA® 35Nfilter or equivalent thereof

Subsequent to nanofiltration, the filtrate may optionally beconcentrated by ultrafiltration and/or the buffer composition adjustedby diafiltration. In certain embodiments, the ultrafiltration is carriedout in a cassette with an open channel screen and the ultrafiltrationmembrane has a nominal molecular weight cut off (NMWCO) of less than ator about 175 kDa or less than at or about 170, 160, 150, 140, 130, 120,110, 100, 90, 80, 70, 60, 50, 40, 30, or fewer kDa. In a preferredembodiment, the ultrafiltration membrane has a NMWCO of no more than 30kDa. In another preferred embodiment, the ultrafiltration membrane has aNMWCO of no more than 30 kDa.

3. Lyophilization and Heat Treatment

In yet other embodiments, the viral activity of a lyophilized ADAMTS13protein (e.g., ADAMTS13) composition, which may have previously beensubjected to other viral inactivation or removal steps such asnanofiltration, may be further reduced by heat treatment of thelyophilized composition. Heat treatments for the inactivation of viralloads in blood factors are well known in the art (for example, see,Piszkiewicz et al., Thromb Res. 1987 Jul. 15;47(2):235-41; Piszkiewiczet al., Curr Stud Hematol Blood Transfus. 1989;(56):44-54; Epstein andFricke, Arch Pathol Lab Med. 1990 March; 114(3):335-40).

VI. ADAMTS COMPOSITIONS

Advantageously, it has been found that ADAMTS proteins, such asADAMTS13, expressed in cell cultures supplemented with zinc, calcium,and/or nicotinamide (vitamin B3) have unexpectedly high specificactivities. As provided herein, methods have been developed for thecontinuous expression and recovery of such ADAMTS proteins with highspecific activities. For example, continuous cell culture methodsprovided herein allow for the expression of greater than 1 mg ADAMTS13per liter per day with specific activities of greater than 1000 U/mg formore than a week or longer. Similarly, methods for reducing the loss ofactivity typically encountered during the purification of ADAMTS13protein are also provided herein.

Accordingly, in one aspect, the present invention provides ADAMTSprotein compositions (e.g., ADAMTS13 compositions) expressed in cellculture according to the methods provided herein. For example, ADAMTSprotein compositions are provided, wherein the protein is expressed byculturing a cell harboring a nucleic acid encoding an ADAMTS protein inculture medium supplemented with at least one component selected fromcalcium, zinc, and nicotinamide (Vitamin B3). Also provided, are ADAMTSprotein compositions (e.g., ADAMTS13 compositions) that are purifiedaccording to a method provided herein. For example, ADAMTS proteincompositions are provided that have been purified according to a methodcomprising a buffer exchange step, wherein the exchange buffer includeszinc and calcium. In preferred embodiments of the invention, the ADAMTSprotein composition is an ADAMTS13 composition.

In another aspect, the present invention provides ADAMTS proteincompositions (e.g., ADAMTS13 compositions) that are prepared by a methodcomprising the expression of the ADAMTS protein in a cell cultureaccording to any method provided herein. In a preferred embodiment, theADAMTS protein is ADAMTS13.

In a related aspect, the present invention provides ADAMTS proteincompositions (e.g., ADAMTS13 compositions) that are prepared by a methodcomprising the inclusion of both zinc and calcium in at least one bufferduring the purification of the ADAMTS protein from a culture medium. Ina preferred embodiment, the ADAMTS protein is ADAMTS13.

The present invention provides ADAMTS protein compositions (e.g.,ADAMTS13 compositions) prepared by a method provided herein. In oneembodiment, the ADAMTS composition (e.g., ADAMTS13 composition) isprepared by culturing a cell harboring a nucleic acid encoding an ADAMTSprotein in culture medium supplemented with at least one componentselected from calcium, zinc, and nicotinamide (Vitamin B3). In aspecific embodiment, an ADAMTS composition is prepared by culturing acell harboring a nucleic acid encoding an ADAMTS protein in culturemedium supplemented with at least two components selected from calcium,zinc, and nicotinamide (Vitamin B3). In yet another embodiment, theculture medium is supplemented with calcium, zinc, and nicotinamide(Vitamin B3). In some embodiments, the culture medium may be an animalprotein-free, an oligopeptide-free, or a chemically defined culturemedium. In a preferred embodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, an ADAMTS protein composition (e.g., ADAMTS13composition) is prepared by culturing a cell harboring a nucleic acidencoding an ADAMTS protein in a culture medium comprising at least at orabout 2 μM zinc. In another embodiment, the composition is prepared byculturing a mammalian cell harboring a nucleic acid encoding an ADAMTSprotein in a culture medium comprising at least at or about 5 μM zinc.In one embodiment, the composition is prepared by culturing a mammaliancell harboring a nucleic acid encoding an ADAMTS protein in a culturemedium comprising at or about between 2 μM and 12 μM zinc. In anotherembodiment, the composition is prepared by culturing a mammalian cellharboring a nucleic acid encoding an ADAMTS protein in a culture mediumcomprising at or about between 5 μM and 12 μM zinc. In yet otherembodiments, the composition is prepared by culturing a mammalian cellharboring a nucleic acid encoding an ADAMTS protein in a culture mediumcomprising at least at or about 2 μM, or at least at or about 3 μM, 4μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15μM, 20 μM, 25 μM, 30 μM, or more zinc. In one embodiment, the culture ismaintained at or about between 35° C. and 37° C. In a specificembodiment, the culture is maintained at or about 36° C. In anotherembodiment, the pH of the culture is maintained at or about between 6.9and 7.3. In one embodiment, the temperature and/or pH of the culture ismaintained for at least 7 days. In one embodiment, the method isperformed under continuous culture conditions. In a specificembodiments, the method is performed under chemostat, turbidostat, orperfusion culture conditions operated in suspension mode. In otherspecific embodiments, the method is performed under chemostat,turbidostat, or perfusion culture conditions operated in adherent mode.In another embodiment, the method is performed under batch cultureconditions. In specific embodiments, the method is performed undersingle-batch, repeated-batch, or fed-batch culture conditions operatedin suspension mode. In other specific embodiments, the method isperformed under single-batch, repeated-batch, or fed-batch cultureconditions operated in adherent mode. In one embodiment, the cellharboring the nucleic acid encoding an ADAMTS protein is a mammaliancell. In particular embodiments, the mammalian cell is a hamster, human,or murine cell. In a specific embodiment, the cell is a CHO cell line,an HEK 293 cell line, or a BHK cell line. In another embodiment, themammalian cell is cultured in an animal protein and/or polypeptide freeculture medium. In yet another embodiment, the mammalian cell iscultured in a synthetic culture medium, which may or may not be free ofanimal proteins and polypeptides. In one embodiment, the culture mediumfurther comprises a polyamine at or about between 0.5 mg/L and 30 mg/L.In another embodiment, the culture medium further comprises a polyamineat or about between 2 mg/L and 8 mg/L. In a specific embodiment, thepolyamine is putrescine. In a preferred embodiment, the ADAMTS proteinis ADAMTS13.

In one embodiment of an ADAMTS protein composition (e.g., ADAMTS13composition), culture medium used for the expression of the ADAMTSprotein may be supplemented with zinc at a final concentration of atleast about 2 μM to at least about 12 μM. In certain embodiments, theculture medium may be supplemented with zinc at a final concentration ofat least about 2 μM, or at least about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM,or higher levels of zinc. Generally, any zinc salt may be used tosupplement the mediums of the invention, non-limiting examples ofacceptable salts include, ZnSO₄.7H₂O, ZnSO₃.2H₂O, (C₆H₅O₇)₂Zn₃.2H₂O,ZnBr₂, ZnBr₂.2H₂O, ZnCl₂, Zn (NO₃)₂.6H₂O, Zn (H₂PO₄)₂.H₂O,(C₂H₃O₂)₂Zn.2H₂O, and the like. In certain embodiments, apharmaceutically acceptable salt of zinc is used to supplement theculture mediums of the invention. In a preferred embodiment, the ADAMTSprotein is ADAMTS13.

In another embodiment, an ADAMTS protein composition (e.g., ADAMTS13composition) is prepared by culturing a cell harboring a nucleic acidencoding an ADAMTS protein in a culture medium comprising at least at orabout 0.5 mM calcium. In another embodiment, an ADAMTS composition isprepared by culturing a mammalian cell harboring a nucleic acid encodingan ADAMTS protein in a culture medium comprising at least at or about1.5 mM calcium. In one embodiment, an ADAMTS composition is prepared byculturing a mammalian cell harboring a nucleic acid encoding an ADAMTSprotein in a culture medium comprising at or about between 0.5 mM and1.5 mM calcium. In yet other embodiments, an ADAMTS composition isprepared by culturing a mammalian cell harboring a nucleic acid encodingan ADAMTS protein in a culture medium comprising at least at or about0.5 mM, or at least at or about 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM,1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM,2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0mM, or more calcium. In one embodiment, the culture is maintained at orabout between 35° C. and 37° C. In a specific embodiment, the culture ismaintained at or about 36° C. In another embodiment, the pH of theculture is maintained at or about between 6.9 and 7.3. In oneembodiment, the temperature and/or pH of the culture is maintained forat least 7 days. In one embodiment, the method is performed undercontinuous culture conditions. In a specific embodiments, the method isperformed under chemostat, turbidostat, or perfusion culture conditionsoperated in suspension mode. In other specific embodiments, the methodis performed under chemostat, turbidostat, or perfusion cultureconditions operated in adherent mode. In another embodiment, the methodis performed under batch culture conditions. In specific embodiments,the method is performed under single-batch, repeated-batch, or fed-batchculture conditions operated in suspension mode. In other specificembodiments, the method is performed under single-batch, repeated-batch,or fed-batch culture conditions operated in adherent mode. In oneembodiment, the cell harboring the nucleic acid encoding an ADAMTSprotein is a mammalian cell. In particular embodiments, the mammaliancell is a hamster, human, or murine cell. In a specific embodiment, thecell is a CHO cell line, an HEK 293 cell line, or a BHK cell line. Inanother embodiment, the mammalian cell is cultured in an animal proteinand/or polypeptide free culture medium. In yet another embodiment, themammalian cell is cultured in a synthetic culture medium, which may ormay not be free of animal proteins and polypeptides. In one embodiment,the culture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine. In a preferredembodiment, the ADAMTS protein is ADAMTS13.

Generally, any calcium salt may be used to supplement the mediums of theinvention. Non-limiting examples of acceptable salts include CaCl₂,CaCl₂, CaFPO₃.2H₂O, CaI₂, CaBr₂, (C₂H₃O)₂)₂Ca, (CHO₂)₂Ca, (C₆H₇O₆)₂Ca,(C₆H₅O₇)₂Ca₃.2H₂O, and the like. In certain embodiments, apharmaceutically acceptable salt of calcium is used to supplement theculture mediums of the invention.

In another embodiment, an ADAMTS protein composition (e.g., ADAMTS13composition) is prepared by culturing a cell harboring a nucleic acidencoding an ADAMTS protein in a culture medium comprising at least at orabout 2 mg/L nicotinamide (vitamin B3). In another embodiment, an ADAMTScomposition is prepared by culturing a mammalian cell harboring anucleic acid encoding an ADAMTS protein in a culture medium comprisingat least at or about 7 mg/L nicotinamide (vitamin B3). In oneembodiment, an ADAMTS composition is prepared by culturing a mammaliancell harboring a nucleic acid encoding an ADAMTS protein in a culturemedium comprising at or about between 2 mg/L and 10 mg/L nicotinamide(vitamin B3). In yet other embodiments, an ADAMTS composition isprepared by culturing a mammalian cell harboring a nucleic acid encodingan ADAMTS protein in a culture medium comprising at least at or about 2mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L, 7 mg/L, 8 mg/L, 9 mg/L, 10 mg/L,15 mg/L, 20 mg/L, or higher concentrations of nicotinamide (vitamin B3).In one embodiment, the culture is maintained at or about between 35° C.and 37° C. In a specific embodiment, the culture is maintained at orabout 36° C. In another embodiment, the pH of the culture is maintainedat or about between 6.9 and 7.3. In one embodiment, the temperatureand/or pH of the culture is maintained for at least 7 days. In oneembodiment, the method is performed under continuous culture conditions.In a specific embodiments, the method is performed under chemostat,turbidostat, or perfusion culture conditions operated in suspensionmode. In other specific embodiments, the method is performed underchemostat, turbidostat, or perfusion culture conditions operated inadherent mode. In another embodiment, the method is performed underbatch culture conditions. In specific embodiments, the method isperformed under single-batch, repeated-batch, or fed-batch cultureconditions operated in suspension mode. In other specific embodiments,the method is performed under single-batch, repeated-batch, or fed-batchculture conditions operated in adherent mode. In one embodiment, thecell harboring the nucleic acid encoding an ADAMTS protein is amammalian cell. In particular embodiments, the mammalian cell is ahamster, human, or murine cell. In a specific embodiment, the cell is aCHO cell line, an HEK 293 cell line, or a BHK cell line. In anotherembodiment, the mammalian cell is cultured in an animal protein and/orpolypeptide free culture medium. In yet another embodiment, themammalian cell is cultured in a synthetic culture medium, which may ormay not be free of animal proteins and polypeptides. In one embodiment,the culture medium further comprises a polyamine at or about between 0.5mg/L and 30 mg/L. In another embodiment, the culture medium furthercomprises a polyamine at or about between 2 mg/L and 8 mg/L. In aspecific embodiment, the polyamine is putrescine. In a preferredembodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, an ADAMTS composition is prepared by culturing a cellharboring a nucleic acid encoding an ADAMTS protein (e.g., ADAMTS13) ina culture medium supplemented with both zinc and calcium. In certainembodiments, the zinc and calcium concentrations can be any of thosedescribed herein. In certain embodiments, the zinc and calciumconcentrations will be one of Var. 94 to Var. 113 (Table 7). In certainembodiments, the culture is maintained for at least 7 days, or at least14 days, 21 days, 28 days, or at least 5 weeks, 6 weeks, 7 weeks, or atleast 2 months, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18 months or longer. In one embodiment, the culture is a perfusionculture. In another embodiment, the culture is a chemostatic culture. Inother embodiments, the culture is a fed-batch or repeated-batch culture.In a preferred embodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, an ADAMTS composition is prepared by culturing a cellharboring a nucleic acid encoding an ADAMTS protein (e.g., ADAMTS13) ina culture medium supplemented with both zinc and nicotinamide (vitaminB3). In certain embodiments, the zinc and nicotinamide (vitamin B3)concentrations can be any of those described herein. In certainembodiments, the zinc and nicotinamide (vitamin B3) concentrations willbe one of Var. 114 to Var. 133 (Table 8). In certain embodiments, theculture is maintained for at least 7 days, or at least 14 days, 21 days,28 days, or at least 5 weeks, 6 weeks, 7 weeks, or at least 2 months, or3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 months orlonger. In one embodiment, the culture is a perfusion culture. Inanother embodiment, the culture is a chemostatic culture. In otherembodiments, the culture is a fed-batch or repeated-batch culture. In apreferred embodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, an ADAMTS composition is prepared by culturing a cellharboring a nucleic acid encoding an ADAMTS protein (e.g., ADAMTS13) ina culture medium supplemented with both calcium and nicotinamide(vitamin B3). In certain embodiments, the calcium and nicotinamide(vitamin B3) concentrations can be any of those described herein. Incertain embodiments, the calcium and nicotinamide (vitamin B3)concentrations will be one of Var. 134 to Var. 149 (Table 9). In certainembodiments, the culture is maintained for at least 7 days, or at least14 days, 21 days, 28 days, or at least 5 weeks, 6 weeks, 7 weeks, or atleast 2 months, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18 months or longer. In one embodiment, the culture is a perfusionculture. In another embodiment, the culture is a chemostatic culture. Inother embodiments, the culture is a fed-batch or repeated-batch culture.In a preferred embodiment, the ADAMTS protein is ADAMTS13.

In one embodiment, an ADAMTS composition is prepared by culturing a cellharboring a nucleic acid encoding an ADAMTS protein (e.g., ADAMTS13) ina culture medium supplemented with zinc, calcium, and nicotinamide(vitamin B3). In certain embodiments, the zinc, calcium, andnicotinamide (vitamin B3) concentrations can be any of those describedherein. In certain embodiments, the zinc, calcium, and nicotinamide(vitamin B3) concentrations will be one of variations Var. 14 to Var. 93(Table 3 to Table 6). In certain embodiments, the culture is maintainedfor at least 7 days, or at least 14 days, 21 days, 28 days, or at least5 weeks, 6 weeks, 7 weeks, or at least 2 months, or 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18 months or longer. In one embodiment,the culture is a perfusion culture. In another embodiment, the cultureis a chemostatic culture. In other embodiments, the culture is afed-batch or repeated-batch culture. In a preferred embodiment, theADAMTS protein is ADAMTS13.

In another embodiment, ADAMTS13 compositions are provided with highspecific activities, for example, a specific activity of at least 600U/mg A13 protein. In another embodiment, the ADAMTS13 composition has aspecific activity of at least 700 U/mg. In another embodiment, theADAMTS13 composition has a specific activity of at least 800 U/mg. Inanother embodiment, the ADAMTS13 composition has a specific activity ofat least 900 U/mg. In another embodiment, the ADAMTS13 composition has aspecific activity of at least 1000 U/mg. In another embodiment, theADAMTS13 composition has a specific activity of at least 1100 U/mg. Inanother embodiment, the ADAMTS13 composition has a specific activity ofat least 1200 U/mg. In another embodiment, the ADAMTS13 composition hasa specific activity of at least 1300 U/mg. In another embodiment, theADAMTS13 composition has a specific activity of at least 1400 U/mg. Inanother embodiment, the ADAMTS13 composition has a specific activity ofat least 1500 U/mg. In another embodiment, the ADAMTS13 composition hasa specific activity of at least 1600 U/mg. In another embodiment, theADAMTS13 composition has a specific activity of at least 1700 U/mg. Inanother embodiment, the ADAMTS13 composition has a specific activity ofat least 1800 U/mg. In another embodiment, the ADAMTS13 composition hasa specific activity of at least 1900 U/mg. In another embodiment, theADAMTS13 composition has a specific activity of at least 2000 U/mg. Inanother embodiment, the ADAMTS13 composition has a specific activity ofat least 2100 U/mg. In another embodiment, the ADAMTS13 composition hasa specific activity of at least 2200 U/mg. In another embodiment, theADAMTS13 composition has a specific activity of at least 2300 U/mg. Inanother embodiment, the ADAMTS13 composition has a specific activity ofat least 2400 U/mg. In another embodiment, the ADAMTS13 composition hasa specific activity of at least 2500 U/mg.

VII. EXAMPLES Example 1

Fed-batch experiments were performed using bioreactor cultures of therecombinant HEK293 cell line 1020/1 013-2 expressing human ADAMTS13 inchemically defined BACD-A13 medium. The effect of supplementing theculture medium with additional zinc was investigated.

Recombinant HEK293 cells expressing human ADAMTS13 were cultivated byfed-batch cell-cultures in 1.5 L bioreactors with blade impellers underan inline controlled pH of 7.20 at 37° C. with a dissolved oxygenconcentration of 20% air saturation. Cell suspension was transferred to2 identical bioreactors containing a DMEM/F12 based chemically definedculture medium (BACD-A13; Table 12). The two cultures were grown inBACD-A13 mediums prepared with either 0.432 mg/L ZnSO₄.7H₂O, as inDMEM/F12, or with an additional supplementation of 1 mg/L ZnSO₄.7H₂O fora final concentration of 1.432 mg/L. The two fed-batch cultures wereotherwise treated the same and thus may be compared directly to oneanother.

The activity of A13 in culture supernatants were measured by FRETS-VWF73assay after centrifuging or filtering the supernatants to removeresidual cells. The total amount of A13 was measured by ELISA, using anA13-specific antibody (Table 10). Supernatant from the culture employingBACD-A13 medium with a standard DMEM/F12 zinc concentration had an A13activity of 728 and 826 mU/mL on days 3 and 6, respectively. Specificactivities for the cultures were 482 mU/μg and 526 mU/μg A13,respectively. In contrast, supernatants from cultures having additionalsupplementation of Zn, while having only slightly improved yields of A13expression (7% and 19% greater at days 3 and 6, respectively), showeddramatically improved total and specific activities A13 activities(Table 11). As can be seen by comparing the results in Table 10 andTable 11, total A13 FRETS-VWF73 activity was 118% higher (1589 vs. 728)at day 3 and 61% higher (1381 vs. 728) at day 6 for cultures grown inthe presence of 1.432 mg/L ZnSO₄.7H₂O as compared to cultures grown at0.432 mg/L ZnSO₄.7H₂O. Similarly, the specific activity of the A13protein in the supplemented cultures was 104% higher (981 vs. 482 mU/μg)at day 3 and 42% higher (739 vs. 526 mU/μg) at day 6.

Samples from the bioreactors were taken and analyzed for A13 by ELISA,A13 activity was measured by FRETS-VWF73 assay. Cell counts weredetermined by Nucleocounter technology. Dilution rates were measured andused for calculation of growth rates and volumetric productivities.

TABLE 10 Fermentation data for fed-batch culture 1 performed in a 1.5 Lbioreactor with BACD-A13 medium without zinc supplementation. Day CellSpecific A13 A13 Specific FRETS A13 Supernatant Concentration GrowthRate FRETS ELISA Activity Yield Yield Harvested [10⁶ Cells/ml] [1/d][mU/ml] [μg/ml] [mU/μg] [U/L/d] [mg/L/d] 3 2.18 0.619 728 1.51 482 2280.41 6 1.88 0.256 826 1.57 526 178 0.32

TABLE 11 Fermentation data for fed-batch experiment performed in a 1.5 Lbioreactor with BACD-A13 medium with zinc supplementation. Day CellSpecific A13 A13 Specific FRETS A13 Supernatant Concentration GrowthRate FRETS ELISA Activity Yield Yield Harvested [10⁶ Cells/ml] [1/d][mU/ml] [μg/ml] [mU/μg] [U/L/d] [mg/L/d] 3 2.48 0.662 1589 1.62 981 5150.45 6 1.38 0.11 1381 1.87 739 249 0.41

TABLE 12 Composition of the chemically defined BACD-A13 cell culturemedium. BACD-A13 Cell Culture Standard concentration Medium Compositionmg/L Amino Acids L-Alanine 13.3500 L-Arginine HCl 147.5000L-Asparagine-H₂O 45.1600 L-Aspartic Acid 19.9500 L-Cysteine HCl-H₂O32.5500 L-Cystine 2HCl 102.3500 L-Glutamic Acid 22.0500 Glycine 26.2500L-Histidine-H₂O HCl 51.4800 L-Isoleucine 74.4700 L-Leucine 119.0500L-Lysine HCl 146.2500 L-Methionine 100.0000 L-Phenylalanine 60.4800L-Proline 63.7400 L-Serine 36.7500 L-Threonine 53.4500 L-Tryptophan29.0100 L-Tyrosine 2Na 2H2O 75.7900 L-Valine 82.8500 salts CalciumChloride (CaCl2) Variable (58.3-174.9) Copper Sulfate (CuSO₄—5H₂O)0.0026 Ferric Nitrate (Fe(NO₃)3—9H₂O) 0.0500 Ferrous Sulfate(FeSO₄—7H₂O) 1.0170 Magnesium Chloride (MgCl2) 28.6400 Magnesium Sulfate(MgSO4) 48.8400 Potassium Chloride (KCl) 311.8000 Sodium Chloride (NaCl)5495.5000 Na2HPO4 Anhydrous 106.5100 NaH2PO4 Anhydrous 54.3500 ZincSulfate Heptahydrate (ZnSO4—27H2O) Variable (0.432-3.432) Sodiumselenite•anhydrous 0.0087 Vitamins Ascorbic Acid 3.499 Biotin 0.0035Choline Chloride 8.980 D-Ca-Pantothenate 2.240 Folic Acid 2.650I-Inositol 12.600 Nicotinamide Variable (2.0-7.0) Pyridoxine HCl 2.031Riboflavin 0.219 Thiamine HCl 2.170 Vitamin B12 0.680 miscellaneousD-Glucose 5000.00 Linoleic Acid 0.042 Lipoic Acid 0.105 Putrescine 2HCl3.681 Thymidine 0.365 Hypoxantine Na 2.390 Sodium Pyruvate 55.000L-Glutamin 1000.0000 Pluronic F68 1000.0000 Ethanolamin 1.5300Na-Hydrogencarbonat 1.5000

Example 2

Fed-batch experiments were performed using bioreactor cultures of therecombinant CHO cell line #938 expressing human ADAMTS13 in chemicallydefined BACD-A13 medium. The effect of supplementing the culture mediumwith additional zinc and/or vitamin B3 was investigated.

Recombinant CHO cells expressing human ADAMTS13 were adapted to achemically defined BCS medium (BCS medium). Clone #938 from DevelopmentWorking Cell Bank#01 (DWCB#01) was thawed and cell inoculum was preparedin BCS medium. Cells were transferred to two 1.5L bioreactors with bladeimpellers and grown under fed-batch cell-culture in proprietary BACD-A13medium with an inline controlled pH of 7.20 at 37° C. with a dissolvedoxygen concentration of 20% air saturation. The two cultures were grownin BACD-A13 mediums prepared with either 0.432 mg/L ZnSO₄.7H₂O, as inDMEM/F12, or with an additional supplementation of 1 mg/L ZnSO₄.7H₂O fora final concentration of 1.432 mg/L. The two fed-batch cultures wereotherwise treated the same and thus may be compared directly to oneanother. Supernatants were harvested at 4 and 7 days and assayed for A13protein and activity levels. After the harvest on day 7, both cultureswere supplemented with an additional 5 mg/L nicotinamide (vitamin B3),for a final concentration of 7.02 mg/L, and grown under identicalconditions for an additional 4 days. Supernatants were again harvestedat day 11 and A13 protein and activity levels were assayed.

In the first culture, grown without zinc supplementation, A13 activitylevels in the harvested supernatant were 642 and 488 mU/m1 on days 4 and7, respectively (Table 13). Specific activities for these supernatantswere 371 and 273 mU/μg on days 4 and 7, respectively. As seen previouslyin example 1, the total and specific activities of the A13 in theculture supernatant supplemented with additional zinc were significantlyincreased (compare Table 13 and Table 14 at harvest days 4 and 7). Asbefore, the total A13 produced in both cultures was similar, however,A13 from the supernatant of the second culture, supplemented withadditional zinc, had 40% and 104% greater total activity at days 4 and7, respectively, as well as 78% and 75% greater specific activity atdays 4 and 7 (Table 14 vs. Table 13). Other parameters, including totalA13 yield and cell growth rate appeared to be unaffected by theadditional zinc supplementation.

After harvesting the supernatants on day 7, both cultures were furthersupplemented with an additional 5 mg/L nicotinamide (vitamin B3) andgrown under identical conditions for an additional 4 days. Supernatantswere harvested as before on day 11. Vitamin B3 supplementation resultedin an increase in the total FRETS-VWF73 activity and the specificactivity of A13 found in the supernatants of both cultures (Table 13 andTable 14, day 11). Notably, these values approximately doubled in bothcultures. At day 11, the supernatant from culture 2, supplemented withaddition zinc and vitamin B3, demonstrated nearly 200% greater (2366 vs.791 mU/mL) total activity and 174% greater (1189 vs. 432 mU/μg) specificactivity than did supernatant harvested from culture 1.

Supplementation of the medium with nicotinamide alone resulted in abouta 60% increase in the total activity (791 mU/ml vs. 488 mU/ml; compareTable 13 at days 7 and 11) and specific activity (432 mU/μg vs. 273mU/μg; compare Table 13 at days 7 and 11) of A13. Similarly,supplementation of the medium with zinc alone resulted in increases ofbetween about 70% and 80% in the total activity and specific activity ofA13 (compare Table 13 and Table 14 at days 4 and 7). Surprisingly,supplementation of the culture medium with both nicotinamide and zincresulted in a synergistic increase of between about 300% to 400% totalactivity (compare Table 14 day 11 with Table 13 days 4 and 7) andbetween about 200% to 300% specific activity (compare Table 14 day 11with Table 13 days 4 and 7) of A13, demonstrating an unexpectedsynergistic interaction between the effects of zinc and nicotinamide.

TABLE 13 Fermentation data for batch experiment CP_07/18_M04: hA13 CHOKlon #985/1 938 DWCB#01. Day Cell Specific A13 A13 Specific FRETS A13Supernatant Concentration Growth Rate FRETS ELISA Activity Yield YieldHarvested [10⁶ Cells/ml] [1/d] [mU/ml] [μg/ml] [mU/μg] [U/L/d] [mg/L/d]4* 2.33 0.539 642 1.73 371 161 0.43 7* 1.58 0.407 488 1.79 273 120 0.4811^(† )  1.65 0.413 791 1.83 432 173 0.37 *BAV-CD-A13; 0.432 mg/LZnSO₄•7H₂O; 2 mg/L nicotinamide ^(†)BAV-CD-A13; 0.432 mg/L ZnSO₄•7H₂O; 7mg/L nicotinamide

TABLE 14 Fermentation data for batch experiment CP_07/18_M07: hA13 CHOKlon #985/1 985 DWCB#01. Day Cell Specific A13 A13 Specific FRETS A13Supernatant Concentration Growth Rate FRETS ELISA Activity Yield YieldHarvested [10⁶ Cells/ml] [1/d] [mU/ml] [μg/ml] [mU/μg] [U/L/d] [mg/L/d]4* 1.73 0.527 898 1.36 660 224 0.34 7* 2.10 0.505 996 2.08 479 252 0.5711^(† )  1.88 0.420 2366 1.99 1189  550* 0.41 *BAV-CD-A13; 1.432 mg/LZnSO₄•7H₂O; 2 mg/L nicotinamide ^(†)BAV-CD-A13; 1.432 mg/L ZnSO₄•7H₂O; 7mg/L nicotinamide

Example 3

A Chemostat cell-culture of the recombinant CHO cell line #640-2expressing human ADAMTS13, was grown in chemically defined BACD-A13medium supplemented with additional zinc and vitamin B3. The 10 Lculture was maintained for 53 days and A13 protein and activityproduction was monitored over time.

Recombinant CHO cells expressing human ADAMTS13 were adapted to achemically defined proprietary medium (BCS medium). A cell bank vial wasthawed and cell inoculum was prepared in BCS medium. Cells propagatedfrom the A13 expression clone #640-2 were transferred to a 10 Lbioreactor with Rushton type impellers and cultivated in repeated batchcultures with proprietary BACD-A13 medium under an inline controlled pHof 7.15-7.20 at 37° C. with a dissolved oxygen concentration of 20% airsaturation. After 2 batch cultures were grown to the final workingvolume of 10 L, the bioreactor was switched to continuous medium feed onday 5 and operated for an additional 48 days in a chemostat mode.

Samples of the supernatant from the bioreactors were taken weekly andanalyzed for A13 protein production by ELISA and A13 activity byFRETS-VWF73 assay. Cell counts were determined by Nucleocountertechnology. Dilution rates were measured and used for calculation ofgrowth rates and volumetric productivities.

Under continuous culture conditions using chemically defined BACD-A13medium supplemented with zinc and nicotinamide at a final concentrationof 1.432 mg/L ZnSO₄.7H₂O and 7.02 mg/L nicotinamide, high levels of A13protein production, between 0.9 and 1.3 mg/L/D, and specific activities,between about 800 and 1100 mU/μg A13, were achieved (Table 15). Theseresults were consistent with protein production and specific activitiesachieved with CHO clone #938, as in example 2. Notably, volumetric andcell specific productivities increased over time in the long termculture, likely due to increasing growth and dilution rates over time.The high specific activity of the expressed A13 could be at leastmaintained at a constantly high level over at least entire 7 weeks theculture was grown under chemostatic conditions. In fact, the specificactivity of the A13 produced in the culture actually increased fromabout 800 mU/μg A13 at week 2 to about 1100 mU/μg A13 at week 7.

TABLE 15 Fermentation data for continuous culture experimentCP_07/30_F02: hA13 CHO Klon #987/1 640-2. Chemostat Cell SpecificDilution A13 A13 Specific FRETS A13 Culture Concentration Growth RateRate FRETS ELISA Activity Yield Yield Week No. [10⁶ Cells/ml] [1/d][1/d] [mU/ml] [μg/ml] [mU/μg] [U/L/d] [mg/L/d] 2 1.43 0.36 0.36 19542.48 788 713 0.91 3 1.56 0.41 0.40 2254 2.32 972 913 0.94 4 1.46 0.380.40 2244 2.41 931 889 0.95 5 1.58 0.43 0.43 2514 2.88 873 1086 1.24 61.70 0.51 0.46 2737 2.71 1010 1270 1.26 7 1.76 0.53 0.52 2322 2.18 10651200 1.13

Example 4

Fed-batch and chemostat cell-culture experiments were performed usingbioreactor cultures of the recombinant CHO cell line #640-2 expressinghuman ADAMTS13 in chemically defined BACD-A13 medium. The effect ofsupplementing the culture medium with different levels of zinc wasinvestigated.

Recombinant CHO cells expressing human ADAMTS13 were adapted to achemically defined proprietary medium, BCS medium, in fed-batch mode.Briefly, DWCB#05 was thawed and cell inoculum was prepared in BCSmedium. Cells were transferred to a 1.5L bioreactors with bladeimpellers and in proprietary BACD-A13 medium supplemented withnicotinamide at a final concentration of 7 mg/L, but without zincsupplementation under an inline controlled pH of 7.20 at 37° C. with adissolved oxygen concentration of 20% air saturation. The batch cellsuspension was then divided into three identical bioreactors containingBACD-A13 medium supplemented with nicotinamide at a final concentrationof 7 mg/L with different Zn supplementations (0 mg/L; 0.5 mg/L; and 1.0mg/L ZnSO₄.7H₂O to a final concentration of 0.432 mg/L; 0.932 mg/L; and1.432 mg/L ZnSO₄.7H₂O).

BACD-A13 mediums included 0.432 mg/L ZnSO₄.7H₂O with or withoutadditional supplementation of zinc. Otherwise, the cultures and mediumwere the same and thus can be compared directly to one another. Cultureswere grown in fed-batch mode for about a week and the supernatant wasassayed for A13 protein production and FRETS-VWF73 activity. All threebioreactors were then switched to chemostat culture mode and operatedfor about 2 weeks under continuous culture conditions using the mediumswith different zinc supplementations, as described above.

Under fed-batch culture conditions, consistent with the results seen inexamples 1 and 2, supplementation of the medium with either 0.5 mg/L or1.0 mg/L ZnSO₄.7H₂O significantly increased the specific activity of A13protein in the supernatant (785 mU/μg and 876 mU/μg, respectively) ascompared to medium not supplemented with additional zinc (437 mU/μg)(Table 16). As before, other parameters, including total A13 proteinproduction and specific cell growth, were unaffected by supplementationwith additional zinc.

TABLE 16 Fermentation data for fed-batch experiments of human A13expression in medium with and without additional zinc supplementation.Cell Specific A13 A13 Specific FRETS A13 Additional Concentration GrowthRate FRETS ELISA Activity Yield Yield Zinc [10⁶ Cells/ml] [1/d] [mU/ml][μg/ml] [mU/μg] [U/L/d] [mg/L/d] 0 1.44 0.420 853.0 1.952 437 230.30.506 0.5 mg/L 1.73 0.502 1687.7 2.151 785 470.0 0.563 1.0 mg/L 1.440.441 1589.2 1.814 876 430.8 0.472

After switching the bioreactors to chemostat mode, the cultures weregrown under continuous culture conditions for about a week (week 2)using the mediums with different zinc supplementations, as describedabove. Samples of the supernatant from the bioreactors were taken weeklyand analyzed for A13 protein production by ELISA and A13 activity byFRETS-VWF73 assay. Similar to the results seen for cultures grown underfed-batch mode, supplementation of the growth medium used for continuousculture growth with additional zinc resulted in the substantialimprovement of A13 specific activities (697 mU/μg and 729 mU/μg for 0.5mg/L and 1.0 mg/L ZnSO4.7H₂O supplementation, respectively) after aweek, as compared to no supplementation (553 mU/μg) (Table 17, Table 18,and Table 19).

Previously, it had been seen that the specific activity of A13 expressedin continuous cell-cultivation actually improved over an extended periodof time (see, example 3). To investigate whether or not this resultcould be repeated, the cultures grown in medium supplemented withadditional zinc were continued for an additional week. As seen in Table18 and Table 19, the specific activity of A13 in the supernatants ofboth cultures again increased over time. The specific activity of A13found in the supernatant of the medium supplemented with 0.5 mg/LZnSO₄.7H₂O increased from 697 mU/mg at week 2 to 759 mU/mg at week 3(Table 18). Similarly, the specific activity of A13 found in thesupernatant of the medium supplemented with 1.0 mg/L ZnSO₄.7H₂Oincreased from 729 mU/mg at week 2 to 812 mU/mg at week 3 (Table 19). Asbefore, specific cell growth was unaffected by supplementation withadditional zinc. Notably, however, A13 protein production appeared toincrease in both cultures supplemented with additional zinc as comparedto production in cultures not supplemented with zinc.

TABLE 17 Fermentation data for chemostat cell-culture experimentswithout additional supplementation of zinc. Chemostat Cell SpecificDilution A13 A13 Specific FRETS A13 Culture Concentration Growth RateRate FRETS ELISA Activity Yield Yield Week No. [10⁶ Cells/ml] [1/d][1/d] [mU/ml] [μg/ml] [mU/μg] [U/L/d] [mg/L/d] 2 1.25 0.340 0.339 1091.81.973 553 370.1 0.669

TABLE 18 Fermentation data for chemostat cell-culture experiments with0.5 mg/L additional supplementation of zinc. Chemostat Cell SpecificDilution A13 A13 Specific FRETS A13 Culture Concentration Growth RateRate FRETS ELISA Activity Yield Yield Week No. [10⁶ Cells/ml] [1/d][1/d] [mU/ml] [μg/ml] [mU/μg] [U/L/d] [mg/L/d] 2 1.37 0.350 0.341 1725.52.476 697 588.4 0.844 3 1.37 0.362 0.353 1971.7 2.597 759 696.0 0.917

TABLE 19 Fermentation data for chemostat cell-culture experiments with1.0 mg/L additional supplementation of zinc. Chemostat Cell SpecificDilution A13 A13 Specific FRETS A13 Culture Concentration Growth RateRate FRETS ELISA Activity Yield Yield Week No. [10⁶ Cells/ml] [1/d][1/d] [mU/ml] [μg/ml] [mU/μg] [U/L/d] [mg/L/d] 2 1.21 0.327 0.340 1681.72.307 729 571.8 0.784 3 1.21 0.330 0.353 1892.2 2.331 812 667.9 0.823

Example 5

Fed-batch and chemostat cell-culture experiments were performed usingbioreactor cultures of the recombinant CHO cell line #F6 expressinghuman ADAMTS13 in chemically defined BACD-A13 medium. The effect ofsupplementing the culture medium with higher levels of zinc wasinvestigated.

Recombinant CHO cells expressing human ADAMTS13 were adapted to achemically defined proprietary medium, BCS medium, in fed-batch mode.The adapted populations were subcloned and a clone #F6 was derivedtherefrom.

Briefly, DWCB#19 was thawed and cell inoculum was prepared in BCSmedium. Cells were transferred to three 1.5L bioreactors with bladeimpellers and in proprietary BACD-A13 medium supplemented with 1.0 mg/L,2.0 mg/L, and 3.0 mg/L ZnSO₄.7H₂O for final concentrations of 1.432mg/L, 2.432 mg/L, and 3.432 mg/L ZnSO₄.7H₂O, respectively. Cultures weregrown under an inline controlled pH of 7.15 at 36° C. with a dissolvedoxygen concentration of 20% air saturation.

Cultures were grown in repeated batch mode (3 batches) for about a weekand the supernatant was assayed for A13 protein production andFRETS-VWF73 activity. All three bioreactors were then switched tochemostat culture mode and operated for 10 days under continuous cultureconditions using the mediums with different zinc supplementations, asdescribed above.

Under fed-batch culture conditions, supplementation of the culturemediums with increasing amounts of zinc further increased the specificactivity of A13 secreted in the culture supernatants. Consistent withthe previous results reported above, supplementation of the medium withan additional 1.0 mg/L ZnSO4.7H₂O resulted in a high specific activityof 806 mU/μg. Further increases in the zinc concentration, i.e.supplementation with 2.0 mg/L and 3.0 mg/L, resulted in even higherlevels of A13 specific activity (880 mU/μg and 889 mU/μg, respectively)(Table 20). As before, other parameters, including total A13 proteinproduction and specific cell growth, were unaffected by supplementationwith additional zinc.

TABLE 20 Fermentation data for batch experiments with additionalsupplementation of zinc (mean data of 3 batches). Cell Specific A13 A13Specific FRETS A13 Additional Concentration Growth Rate FRETS ELISAActivity Yield Yield Zinc [10⁶ Cells/ml] [1/d] [mU/ml] [μg/ml] [mU/μg][U/L/d] [mg/L/d] 1.0 mg/L 1.78 0.592 1666.6 2.074 806 633.8 0.687 2.0mg/L 1.68 0.596 1678.0 1.883 880 621.8 0.619 3.0 mg/L 1.55 0.589 1670.11.844 889 580.8 0.625

After switching the bioreactors to chemostat mode, the cultures weregrown under continuous culture conditions for 10 days using the mediumswith different zinc supplementations, as described above. Samples of thesupernatant from the bioreactors were analyzed for A13 proteinproduction by ELISA and A13 activity by FRETS-VWF73 assay. Unlike theresults seen for cultures grown under fed-batch mode, supplementation ofthe growth medium used for continuous culture growth with higher levelsof zinc resulted in a decrease in the specific growth rate and overallcell viability. The chemostat culture grown in medium supplemented withan additional 1.0 mg/L ZnSO4.7H₂O continued to display high cellviability (88.6%) and a specific growth rate (0.256 per day) (Table 21)consistent with the previous results described above.

In contrast, cultures grown in mediums supplemented with higher levelsof ZnSO₄.7H₂O, 2.0 mg/L and 3.0 mg/L, displayed significant reductionsin the levels of cell viability (72.1% and 80.4%, respectively) andspecific growth rates (0.091 and 0.134 per day, respectively). Notably,however, the specific activity of A13 in the two culture supernatantsremained high (863 mU/μg and 771 mU/μg, respectively) (Table 21).

TABLE 21 Fermentation data for chemostat cell-culture experiments withadditional supplementation of zinc. Cell Specific Dilution Cell A13 A13Specific FRETS A13 Glucose Additional Concentration Growth Rate RateViability FRETS ELISA Activity Yield Yield Consumption Zinc [10⁶Cells/ml] [1/d] [1/d] [%] [mU/ml] [μg/ml] [mU/μg] [U/L/d] [mg/L/d][g/L/d] 1.0 mg/L 1.28 0.256 0.336 88.6 2716.0 3.127 868 911.5 1.050 1.212.0 mg/L 0.55 0.091 0.321 72.1 1408.9 1.632 863 452.7 0.524 0.58 3.0mg/L 0.56 0.134 0.321 80.4 1359.8 1.763 771 436.5 0.566 0.64

Example 6

Human A13 protein was expressed in the recombinant CHO cell line #640-2in a chemically defined BACD-A13 medium supplemented with additionalzinc and nicotinamide in 50 L bioreactors. After harvesting thesupernatants of the cultures, a comparison of Ultra/Diafiltration (50kD) steps using buffers with and without zinc and calcium was performed.

Recombinant CHO cells expressing human ADAMTS13 were adapted to achemically defined proprietary medium, BCS medium. Briefly, a DWCB wasthawed and cell inoculum was prepared in BCS medium. Cells weretransferred via 10 L a bioreactor to a 50 L bioreactor with Rushton typeimpellers and cultivated in chemostat mode in proprietary BACD-A13medium under an inline controlled pH of 7.15-7.20 at 37° C. with adissolved oxygen concentration of 20% air saturation.

Supernatants from the bioreactors were filtered and cell-free harvestswere ultrafiltrated using a 50 kD PES membrane (concentration factorapproximately 1:10) and then diafiltered with 5 volumes of a buffercontaining 50 mM NaCl and 20 mM TRIS at pH 7.7. Diafiltration bufferwith or without 2 mM Ca and 5 μM Zn was compared to determine the effecton activity loss between diafiltration and chromatographic purification.

Specific activities, recorded as mU FRETS-VWF73 per μg A13 detected byELISA, were determined immediately after diafiltration and right beforeloading the sample for further purification. The samples were stored atbetween about 2 and 8° C. for a maximum time of 3 days (about less than80 hours). When samples were stored for longer periods of time, theywere kept at less than −15° C. between end of diafiltration andbeginning of chromatography. Samples were measured and comparedimmediately after diafiltration and right before loading thechromatographic column.

Despite the relatively short hold time between the ultra/diafiltrationsteps and additional purification steps, a significant loss of A13specific activity (23.3%) occurred when the diafiltration buffer lackedzinc and calcium. In contrast, when the diafiltration buffer contained 2mM Ca and 5 μM Zn was used, the loss of A13 specific activity (7.3%) wasgreatly reduced (Table 22).

TABLE 22 Results of diafiltration experiments using diafiltration bufferwith and without calcium and zinc. Specific Specific Activity inActivity After Purification % Loss Sample Diafiltration Filtration Loadin Activity 1 w/o Ca/Zn 999 740 25.9 2 w/o Ca/Zn 924 734 20.6 Mean w/oCa/Zn 962 737 23.3 Std. dev w/o Ca/Zn 53 4.2 N/A 3 with Ca/Zn 849 8351.6 4 with Ca/Zn 951 985 −3.6 5 with Ca/Zn 929 877 5.6 6 with Ca/Zn 1131844 25.4 Mean with Ca/Zn 965 885 7.3 Std. dev with Ca/Zn 119 68.9 N/A

Example 7

Chemostat and perfusion continuous cell-culture methods were compared todetermine the relative production and specific activities of A13expressed in several recombinant CHO cell lines.

Briefly, different clones of recombinant CHO cell expressing recombinanthuman A13 were adapted to a chemically defined proprietary medium (BCS),which was used for inoculums preparation. Cells were further cultivatedin bioreactor cultures in proprietary BACD-A13 medium supplemented withzinc and nicotinamide at final concentrations of 1.432 mg/L ZnSO₄.7H₂Oand 7.02 mg/L nicotinamide. Inocula from each cell clone were thentransferred to two bioreactors, of which one was cultured underchemostatic conditions (CST) and the other under perfusion, as indicatedin Table 23. Cells were cultured at a density of between about 2×10⁶ and4×10⁶ cells/ml on Cytopore™ II microcarriers (GE Biosciences).

Cultures were grown under continuous culture conditions for 3 to 4 weeksand samples of the supernatant from the bioreactors were analyzedperiodically for A13 protein production by ELISA and A13 activity byFRETS-VWF73 assay. Values given in Table 23 represent averages for the 3to 4 week period. Consistent with results obtained in examples 1 to 6above, cultures grown under chemostatic produced about 1 mg/L/d to about2 mg/L/d of A13 protein having high specific activities of about 700mU/μg to about 1000 mU/μg (Table 23). Strikingly, cultures grown underperfusion culture conditions consistently demonstrated higher proteinproduction and activity levels of A13 in the culture supernatant thandid the identical clones grown under chemostatic conditions. Specificactivities for A13 produced in the perfusion cultures were very high,with three of the four clones demonstrating at least about 1000 mU/μg.

TABLE 23 Results from experiments comparing chemostat and perfusioncell-cultivation. Specific FRETS A13 Volume activity Yield Yield Clone[L] Mode [mU/μg] [U/L/d] [mg/L/d] F6 50.0 CST 800 800 1.0 F6 10.0Perfusion 1000 1600 1.6 D6 1.5 CST 690 900 1.3 D6 1.5 Perfusion 890 17001.9 X1 1.5 CST 1040 2400 2.3 X1 1.5 Perfusion 1000 2600 2.6 E2 1.5 CST710 500 0.7 E2 1.5 Perfusion 1000 1600 1.6

Example 8

In order to determine the effect of temperature and pH on the productionof recombinant human ADAMTS13 protein grown in eukaryotic cell cultureusing a chemically defined animal protein-free culture medium, cultureswere grown at temperatures between 35.0° C. and 38.0° C. at pH's ofbetween 7.05 and 7.30.

Briefly, recombinant CHO cells expressing human A13 were transferred to1.5 L bioreactors with blade impellers, in proprietary BACD-A13 medium.Cultures were grown under inline controlled pH's between 7.05 and 7.30at temperatures ranging from 35.0° C. to 38.0° C. with a dissolvedoxygen concentration of 20% air saturation. The experiments weredesigned and executed using the response surface approach. Thestatistical analysis was done using the statistical Software packageMinitab® 15.1.0.0.

Samples from the bioreactors were taken and analyzed for A13 by ELISA,A13 activity was measured by FRETS-VWF73 assay. Cell counts weredetermined by Nucleocounter technology. Dilution rates were measured andused for calculation of growth rates and volumetric productivities.

As can be seen in FIGS. 1 and 2, both temperature and pH had asubstantial effect on the volumetric A13 productivity (measured byFRETS-VWF73, FIG. 1), and specific activity (FRETS-VWF73activity/antigen by ELISA, FIG. 2). Specifically, maximal volumetricFRETS productivity was achieved in cultures grown in a lower pH rangebetween about 7.05 and 7.2, especially about 7.10 and temperaturesbetween about 35.0° C. and 37.0° C., especially about 36° C.Specifically, maximal specific activity (FRETS-VWF73 to antigen byELISA) was achieved in cultures grown in a lower pH range between about7.05 and 7.2, especially below 7.10 and temperatures between about 35.0°C. and 37.0° C., especially below 36° C. Specifically optimal conditionsfor A13 production were achieved with a combination of a temperature ofabout 36° C. and a pH of about 7.10 in terms of optimal yield andproduct quality.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1-21. (canceled)
 22. A method for expressing a disintegrin andmetalloproteinase with thrombospondin motifs (ADAMTS) protein, themethod comprising culturing, by continuous cultivation, a mammalian cellharboring a nucleic acid encoding an ADAMTS protein in a culture mediumcomprising at least 3 μM zinc, wherein the continuous cultivationincludes maintaining a cell density of from 1×10⁶ cells/mL to 4×10⁶cells/mL for at least seven days.
 23. The method of claim 22, whereinthe culture medium comprises from 3 μM to 12 μM zinc.
 24. The method ofclaim 22, wherein the culture medium comprises about 5 μM zinc.
 25. Themethod of claim 22, wherein the culture medium further comprises from0.5 mM to 1.5 mM calcium.
 26. The method of claim 23, wherein theculture medium further comprises from 0.5 mM to 1.5 mM calcium.
 27. Themethod of claim 24, wherein the culture medium further comprises from0.5 mM to 1.5 mM calcium.
 28. The method of claim 22, wherein themammalian cell is selected from the group consisting of a CHO cell, aBHK cell, and an HEK cell.
 29. The method of claim 23, wherein themammalian cell is selected from the group consisting of a CHO cell, aBHK cell, and an HEK cell.
 30. The method of claim 24, wherein themammalian cell is selected from the group consisting of a CHO cell, aBHK cell, and an HEK cell.
 31. The method of claim 22, wherein thecontinuous cultivation is chemostatic cell cultivation.
 32. The methodof claim 23, wherein the continuous cultivation is chemostatic cellcultivation.
 33. The method of claim 24, wherein the continuouscultivation is chemostatic cell cultivation.
 34. The method of claim 29,wherein the continuous cultivation is chemostatic cell cultivation. 35.The method of claim 22, wherein the ADAMTS13 protein is a human ADAMTS13protein.
 36. The method of claim 23, wherein the ADAMTS13 protein is ahuman ADAMTS13 protein.
 37. The method of claim 24, wherein the ADAMTS13protein is a human ADAMTS13 protein.
 38. The method of claim 29, whereinthe ADAMTS13 protein is a human ADAMTS13 protein.
 38. The method ofclaim 31, wherein the ADAMTS13 protein is a human ADAMTS13 protein. 40.The method of claim 32, wherein the ADAMTS13 protein is a human ADAMTS13protein.
 41. The method of claim 34, wherein the ADAMTS13 protein is ahuman ADAMTS13 protein.